Phenotypic and genetic stepwise changes in Staphylococcus aureus during in vitro 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 Staphylococcus aureus. This study aims to develop higher-level tigecycline-resistant S. aureus mutants (TRSAms) and explore the mechanisms behind decreasing susceptibility to tigecycline. In this study, S. aureus 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 in vivo and in vitro. The phenotypes of fitness cost showed significant differences in these high-level TRSAms. Whole-genome sequencing analysis detected synchronous mutations between yycH and fakA repeatedly in three high-level TRSAms from different parent strains. Further cloning experiments demonstrated that the complementary yycH gene increased susceptibility to tigecycline in TRSAms, and deletion mutant construction and complementation of Glu283Ter YycH confirm its critical role in tigecycline susceptibility in S. aureus. We also scanned the global genome to evaluate clinical importance; mutations on rpsJ 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 S. aureus. It is considered that the yycH gene has been involved in decreasing tigecycline susceptibility in S. aureus.