## 1 Introduction  
_Staphylococcus aureus_ is an important Gram-positive pathogen that causes major health problems, especially in the form of recalcitrant infections [1]. Typical examples are infective endocarditis, osteomyelitis, skin and soft tissue infections, and medical device-related infections. These conditions are particularly difficult to manage due to the timely expression of specific virulence factors and the biofilm growth mode. Biofilm formation is an important mechanism employed by bacteria, including _S. aureus_, to sequester scarce nutrients and evade host defenses and antimicrobial chemotherapy [2].  
This growth mode is characterized by the aggregation of bacteria into microcolonies, which become engulfed within a primarily self-produced matrix consisting of proteins, extracellular DNA (eDNA), carbohydrate polymers, and/or even host-derived biomolecules. Similar to other biofilm-forming bacteria, _S. aureus_ also produces many adhesive proteins that facilitate the attachment of the cells onto host tissue and abiotic surfaces [3; 4; 5]. In addition to providing mechanical protection and enabling the efficient use of nutrients, the physico-chemically diverse biofilm architecture gives rise to phenotypic heterogeneity within the inhabiting cells [6; 7], creating optimal conditions for acquisition of a highly tolerant state known as persistence. Persisters are nongrowing, transiently antibiotic-tolerant bacteria surviving exposure to multiple stresses without undergoing genetic change, which besides in biofilm populations are also present in planktonic cultures [8; 9]. Bactericidal antibiotics quickly kill most of a bacterial population, leaving a small fraction of cells that survive by entering a persister state; however, the presence of such survivors is not considered when initiating treatment [10].  
Recent studies established that conditions interfering with transcription, translation, or ATP synthesis dramatically increase the persister frequency from 0.01% to 10%-100% [11]. In particular, the cellular depletion of ATP has been found to be a key event for triggering persister formation in _S. aureus_, in which neither the stringent response nor the TA (toxin-antitoxin) system were involved [8]. The cell surface-associated proteins, or surfaceome, are expected to play a role when cells switch into a persister state, as they form the first line of molecular interaction with changes of the environment. To date, the majority of the systematic studies investigating the surfaceome dynamics in _S. aureus_ have been conducted with exponential or stationary phase planktonic cells, but cells cultured for prolonged time periods in either planktonic or biofilm states have not been systematically explored by proteomics. As tolerance characteristics outside the immune-evasive barrier are similar among stationary-phase bacteria, planktonic persisters, and biofilm cells [6], we investigated the growth mode-dependent surfaceome changes on _S. aureus_ ATCC25923, a control strain used for susceptibility testing [12]. To this end, the cells were prepared in a way that would enhance the formation of persisters during both the planktonic and biofilm growth. As the phenotypic state of the bacterial inoculum can also have a remarkable effect on the resulting phenotypic features of the bacterial cell [13; 14], we also investigated whether the physiological stage of the cells preceding biofilm growth affects the surfaceomes in aging biofilms. To the best of our knowledge, this study is among the first to describe and compare surfaceomes from _S. aureus_ cultures grown for prolonged time periods in planktonic and biofilm forms.