## 5 Conclusions  
The present study revealed both the growth mode- and growth stage-dependent surfaceome changes in levofloxacin-tolerant _S. aureus_ ATCC25923 cells grown for prolonged time periods. The resistant planktonic cells were predicted to enhance cell wall strengthening activities and proteolysis/ peptidolysis to release amino acids for growth or development of persistence during prolonged growth. Maturing biofilms were suggested to exploit distinct mechanisms, involving the immune evasion, cold-shock, DNA, and/or RNA binding activities, for increasing resistance and persistence. The TCA-cycle, the CidC-dependent cell death, and ROS scavenging activities exhibited similar abundance trends during both growth modes, implying that these pathways could promote the viability by diverting the carbon flux towards neutral rather than acetate end products and/or by generating beneficial mutations. It could be that the surfaceome changes involving specific moonlighters do not directly correlate with their protein synthesis changes in the cytoplasm. For example, the key TCA enzymes, AcnA and 2-OGDH, could have been increasingly exported from the cells to maintain proper NADH/NAD balance for preventing ROS-mediated cell death. While exporting certain cytoplasmic proteins out of the cells could be an energetically favorable way for _S. aureus_ to improve viability under hostile conditions, further studies are needed to confirm this possibility. Nevertheless, cells from biofilm cultures initiated with stationary-phase cells exhibited higher in vitro viability than cells from biofilms initiated with exponential cells. We also demonstrated that exponentially growing planktonic cells are more adherent to hydrophobic surfaces than stationary phase cells that were more adherent to human fibronectin. This indicates that the physiological state of the cells prior to biofilm formation plays a role in clinical settings. In addition, our study demonstrated that the phenotypic state of the planktonic cells prior to adherent growth can have a great impact on the viability of the cells, thereby contributing to persistence. These findings together with the identification results suggest that both classical surface proteins and moonlighters are needed to increase the immune-evasive and persistent traits in _S. aureus_. Taken together, this study is among the first reports describing growth mode-dependent surfaceome changes in aging _S. aureus_ cells, demonstrating that distinct metabolic activities are exploited in biofilm and planktonic cells to achieve resistance. **Supplementary Materials:** The following are available online at [[http://www.mdpi.com/2076-2607/8/1/106/s1](http://www.mdpi.com/2076-2607/8/1/106/s1)]([http://www.mdpi.com/2076-2607/8/1/106/s1](http://www.mdpi.com/2076-2607/8/1/106/s1)). Figure S1: Distribution of the identified proteins in different replicate samples. Figure S2: Distribution of the proteins associated with clusters 1-6 (heatmap shown in Figure 5) as a function of their GO terms. Figure S3: Abundances of the fadABDEX (A) and dnaJ-dnaK-grPE (B) operon-coded proteins in planktonic and biofilm cells at the indicated time points. Table S1: The MaxQuant output-file showing the identified proteins after searching the MS/MS raw data against the Staphylococcus aureus ATCC25923 protein database. Table S2: List of all predicted and identified surfaceome proteins from planktonic and biofilm cells of Staphylococcus aureus ATCC25923. The LCP intensities of the identified proteins from the MaxQuant analysis were imported and transformed to loga average, and the protein quantification and calculation of statistical significance was carried out using Perseus. Proteome bioinformatics was conducted for each protein to uncover their physico-chemical properties (pl, Mv, pl, GRAVY), the presence of classical- and non-classical secretion motifs (SecP, SignalP4.1 and TatP), TMDs (TMHMM), lipobox and subcellular location and function (ScanProsite, GO annotations). Low abundance proteins, green. High abundance proteins, red.