Background: Staphylococcus pseudintermedius is a major opportunistic pathogen of dogs and the primary cause of canine pyoderma and other infections. The global emergence of methicillin-resistant S. pseudintermedius (MRSP) represents a significant challenge in veterinary medicine due to its frequent multidrug-resistant phenotype and limited therapeutic options. Methods: We describe the phenotypic and genomic characterization of an MRSP isolate recovered from a vaginal swab of an 11-year-old dog diagnosed with vulvovaginitis in southern Italy. Antimicrobial susceptibility testing was performed using broth microdilution according to CLSI VET01S guidelines. Whole-genome sequencing was conducted to determine sequence type and antimicrobial resistance determinants. Results: The isolate was identified as S. pseudintermedius by MALDI-TOF MS and confirmed by genomic analysis. Multilocus sequence typing assigned the strain to sequence type ST2333. Phenotypically, the isolate exhibited multidrug resistance, including resistance to β-lactams, macrolides, lincosamides, tetracyclines, aminoglycosides, fluoroquinolones, and trimethoprim–sulfamethoxazole, while remaining susceptible to amikacin, rifampicin, florfenicol, and vancomycin. Whole-genome sequencing confirmed the presence of mecA and additional resistance determinants consistent with the observed phenotype. Conclusions: This report suggests the possible occurrence of an MDR MRSP ST2333 lineage in southern Italy and highlights the importance of combined phenotypic and genomic surveillance to support antimicrobial stewardship in veterinary medicine within a One Health framework.
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