Phenotypic and genetic stepwise changes in <i>Staphylococcus aureus</i> during <i>in vitro</i> adaptive laboratory evolution under the selective pressure of tigecycline

Compared with tigecycline-resistant gram-negative bacteria, tigecycline adaptive laboratory evolution (ALE) has been preliminarily performed in <i>Staphylococcus aureus</i>. This study aims to develop higher-level tigecycline-resistant <i>S. aureus</i> mutants (TRSAms) and explore the mechanisms behind decreasing susceptibility to tigecycline. In this study, <i>S. aureus</i> strains were cultured in serial-increasing concentrations of tigecycline and successfully obtained high-level TRSAms. Different phenotypic changes in high-level TRSAms were assessed by growth rate measurement, autolysis assays, mutant frequency determination, and virulence evaluations <i>in vivo</i> and <i>in vitro</i>. The phenotypes of fitness cost showed significant differences in these high-level TRSAms. Whole-genome sequencing analysis detected synchronous mutations between <i>yycH</i> and <i>fakA</i> repeatedly in three high-level TRSAms from different parent strains. Further cloning experiments demonstrated that the complementary <i>yycH</i> gene increased susceptibility to tigecycline in TRSAms, and deletion mutant construction and complementation of Glu283Ter YycH confirm its critical role in tigecycline susceptibility in <i>S. aureus</i>. We also scanned the global genome to evaluate clinical importance; mutations on <i>rpsJ</i> detected in this study are associated with the MRSA ST5-t002 isolates and omadacycline selective mutants. In summary, we described a complete trajectory of phenotypic and genotypic changes in the ALE process for decreasing susceptibility to tigecycline in <i>S. aureus</i>. It is considered that the <i>yycH</i> gene has been involved in decreasing tigecycline susceptibility in <i>S. aureus</i>.