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Description: Staphylococcus pseudintermedius is a urease-producing bacteria which is a major cause of magnesium ammonium phosphate (MAP) urolithiasis in canine. A positive urolith culture is an important risk factor for MAP urolithiasis in canine. The mechanism underlying the metabolic changes of S. pseudintermedius after crystallization in artificial urine (AU) needs more defined baseline metabolic information. Therefore, we extensively investigated the metabolic changes of S. pseudintermedius extensively after crystallization in AU. A high urease activity and positive biofilm formation strain, entitled the S. pseudintermedius (SPMAP09) strain, was isolated from canine MAP uroliths, and analyzed using nuclear magnetic resonance (NMR) spectroscopy-based metabolomics. The molecular mechanism-specific metabolic phenotypes were clearly observed after crystallization in AU at day 3. The crystals induced by SPMAP09 were also confirmed and the major chemical composition identified as struvite. Interestingly, our findings demonstrated that a total of 11 identified metabolites were significantly changed. The levels of formate, homocarnosine, tyrosine, cis-aconitate, glycolate, ethyl malonate, valine and acetate level were significantly higher, accompanied with decreased levels of inosine, glucose, and threonine at day 3 compared with the initial time-point (day 0). In addition, our results exhibited that the glyoxylate and dicarboxylate metabolism was significantly related to the SPMAP09 strain at day 3 in AU. Thus, metabolic changes of the SPMAP09 strain after crystallization in AU potentially helps to explain the preliminary molecular mechanism for the crystals induced by S. pseudintermedius.