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Staphylococcus aureus ( S. aureus ) is a clinical pathogen of great significance causing metastatic or complicated infections. ST5 clonotype isolates have dominated S. aureus infections for more than 10 years in Shanghai, China, and the proportion of methicillin-susceptible S. aureus (MSSA) has remarkably increased in the past decades. By whole-genome sequencing (WGS) 121 ST5 clonotype S. aureus isolates using next-generation sequencing (NGS) platforms and characterizing the evolutionary dynamics of ST5 linages, we found that MSSA evolved independently, making it a subtype differed from other MRSA clones. Drug resistance gene analysis by using the NGS data demonstrated that ST5 clonotype MRSA might be more tolerant under the threat of antimicrobials, which was confirmed in further in vitro susceptibility tests. However, MSSA subtype isolates exhibited relatively high virulence upon the analysis of virulence factors. Furthermore, MSSA subtype isolates displayed higher hemolysis capacity and higher ability to adhere to epithelial cells including A549 human alveolar epithelial cells and HaCaT human skin keratinocytes, caused more severe infections in murine abscess model. With its high virulence and enhanced magnitude in the past decades, the ST5 MSSA subtype poses a serious clinical threat hence more attention should be paid to the prevention and control.

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