1H NMR- and GC-MS-based metabolomics reveal the metabolic response of Pseudomonas granadensis to the presence of Staphylococcus epidermidis.

Soil hosts a myriad of highly competitive microorganisms that develop specialized metabolic strategies to survive and interact with competitors. In this study, we report that a soil strain of Pseudomonas granadensis isolated from the Campi Flegrei volcanic area (Southern Italy) inhibits the human pathogen Staphylococcus epidermidis through cell free supernatant (CFS) derived from mixed cultures. Interestingly, this inhibitory activity emerged only in CFSs obtained from mixed cultures of the two species. CFSs from P. granadensis monocultures showed no activity, indicating that the presence of S. epidermidis triggers a specific metabolic response by P. granadensis. To investigate this phenomenon, we applied an integrated metabolomics strategy combining 1H NMR and GC-MS. This dual-platform approach revealed extensive metabolic reprogramming of P. granadensis when cultured in the presence of S. epidermidis. The metabolic shift included enhanced glucose consumption and increased production of lactic, acetic, succinic, fumaric, and gluconic acids, together with multiple fatty and dicarboxylic acids. These changes indicate activation of stress-related metabolic pathways, including increased flux through the Entner-Doudoroff pathway, induction of the glyoxylate shunt of the TCA cycle, and stimulation of fatty acid biosynthesis. Overall, this study highlights the value of metabolic profiling for exploring microbial strategies in competitive environments and provides insight into the ability of soil bacteria to generate antimicrobial metabolites in response to competitive pressure.