AbstractVancomycin has been the primary agent used to treat serious Methicillin-resistant Staphylococcus aureus (MRSA) infection for many years. However, the rise of MRSA infection rates and the extensive use of vancomycin have led to the emergence of reduced vancomycin susceptibility. Therefore, four typical Staphylococcus aureus (S. aureus) strains from different clinical specimens were derivated by vancomycin in vitro to better clarify their phenotypic and molecular characteristics. Some experiments, such as stepwise selection of vancomycin-resistant strains, pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility test, population analysis profile-area under the curve (PAP-AUC), molecular typing, transmission electron microscopy, δ-hemolysin expression, autolysis assay, biofilm assay and quantitative real-time polymerase chain reaction (qPCR) for gene expression were carried out to compare the derivated bacteria with their parental strains. Results showed that the observed phenotypes of vancomycin-resistant strains such as hemolysin, autolysis and biofilm significantly reduced, which were associated with vancomycin resistance capability of the selected strain. The changes of phenotype and regulatory genes expression were inversely proportional to the vancomycin minimum inhibitory concentration (MICvan). Most heterogeneous vancomycin intermediate Staphylococcus aureus (hVISA) or VISA strains belonged to spa type t570 and agr group II. In summary, the clinical isolated vancomycin susceptible Staphylococcus aureus (VSSA), hVISA and VISA could be derivated into high vancomycin-resistant VISA in vitro, but it was difficult for them to develop into vancomycin resistant Staphylococcus aureus (VRSA). VISA and hVISA could gradually adapt to the environment with the vancomycin concentration that continuously elevates.

The extensive use of vancomycin has led to the development of MRSA ( methicillin- resistant Staphylococcus aureus ) strains with varying degrees of resistance to vancomycin. Accumulation of surplus cell wall material, decreased cross-linking of peptidoglycan, and/ or other cell wall alterations has been put forward to explain the VISA phenotype. To our knowledge, the protein profiles of hVISA and VISA strains are rarely analyzed via quantitative comparative proteomics. In this study, we subjected subcellular fractions isolated from two isogenic S. aureus strains ( vancomycin-intermediate resistant S. aureus ) to proteomic analysis, using an integrated quantitative proteomic approach assisted by bioinformatic analysis. In total,128 up-regulated proteins ( mainly AhpC, Adh, ArgF et al.) were identified,21 down-regulated proteins ( mainly LtaS, SdrD, MsrR, MsrB, OatA et al.) were obtained. The largest group of differentially expressed proteins is composed of enzymic proteins associated with metabolic and catalytic activity, which accounts for 50% and 51% of the total proteins, respectively.Some proteins which take an indispensable part in the regulatory networks of S. aureus with vancomycin treatment are related to Cell wall metabolism ( MurA ) , Cell adhesion ( SdrC, SdrD, ClfA , ClfB ) , Proteolysis ( Atl, LytM, SceD ) and Pressure response ( MsrA , MsrB, AhpC ) process.In conclusion, our proteomic study revealed regulatory proteins associated with vancomycin resistance in S. aureus, and some of these proteins are involved in the regulation of cell metabolism and function, providing protential targets for further development of drug resistance strategies.