Monitoring how the metabolic behavior of mycoplasmas is affected by the nutrients: a proof-of-concept demonstration on Mycoplasma gallisepticum by DART-HRMS

Mycoplasmas are known for being fastidious, with scientists still struggling to propagate them in vitro. Improving their culture is vital for future research, despite limited metabolomics studied available. This study explored the chemical composition changes in four liquid media (A, B, C, D) inoculated with Mycoplasma gallisepticum (MG), aiming to uncover overlooked metabolic features. We used Direct Analysis in Real Time High-Resolution Mass Spectrometry (DART-HRMS) across five time points over 71 hours. Integration of DART-HRMS data with statistical analysis showed that media A and D initially contained higher glucose levels, which declined over time. Lactic acid levels rose in all media, with signals reaching saturation at the latest time points. Subsequent pathway enrichment analysis revealed an unexpected overexpression of arginine metabolism. Spermidine accumulation in certain media suggests a potential link to inhibited biofilm formation, opening questions about polyamine function in MG. Histidinal accumulation indicated an unpredicted amino acid synthesis capability in a mycoplasma and MG’s inability to convert arginine into glutamic acid. Additionally, MG was observed to utilize creatine when present. These findings highlight the importance of metabolomics in understanding enigmatic microorganisms like mycoplasmas, reaffirming that environmental factors drive alternative metabolic routes in MG and opening new research avenues.