## 1 Introduction
### S. aureus in Biofilms and Resistance and Tolerance Mechanisms
There remains little agreement on the exact resistance or tolerance mechanisms of biofilms against antibacterial agents [64; 91]. There are several reasons why biofilms have such a heightened resistance or tolerance against antibacterial agents: (i) the slow and impaired diffusional penetration of antibacterial agents across the physical barrier (the matrix and layers of cells) of the biofilms [1; 64; 65; 67; 71]. This barrier is also a protective hurdle against the invasion of infected hosts' immune macrophages into biofilms [92]; (ii) the existence of persister cells and small-colony variants (SCVs, characterized by slow growth, reduced metabolism, less motility, and associated with environmental stress, such as antibiotic treatment causing their phenotypic or genetic changes) that are tolerant and resistant to antibiotics, respectively [1; 57; 70; 71; 93; 94; 95]; (iii) stress-adaptive responses of bacterial cells in the biofilm and the induction of _rpoS_ (RNA polymerase sigma factor)-mediated stress cause their slow growth and protection against environmental stressors and chemical agents [1; 64; 96]; (iv) heterogeneity of the chemical microenvironment (e.g., oxygen levels and pH gradients, ranging from <5.5 to \(\approx\)6.5 [97; 98]) within the biofilm antagonize antibacterial agents [1; 54; 56; 65; 94]; (v) upregulation of resistance-associated genes in biofilms [1; 56]; (vi) uptake of resistance genes through horizontal gene transfer in biofilms, i.e., plasmid conjugation and mobilization [58; 71]; and (vii) the multispecies bacterial communities within the biofilm consortia [56; 70]. Figure 1: Nanotheranostics diagnose via visual detection, or fluorescence microscope, or monitoring. Nanotheranostics treat via, e.g., photothermal therapy (PTT) with gold nanoparticles (NPs) (GNPs) inducing thermal damage. Photodynamic therapy (PDT) releasing reactive oxygen species (ROS). Physicochemical properties of NPs as metal ions release (MIR), their cargo (e.g., antibiotics), and physical disruption by nanoknives or structurally nanoengineered antimicrobial peptide polymers (SNAPPs). “Multifunctional theranostic nanofibers” could be directly electrospun on a diabetic foot ulcer. _Staphylococcus aureus_ associated implant (hip or breast) and wound infections. Steps of _S. aureus_ biofilm formation on the hip implant are initial adhesion, microcolonies formation within an extracellular polymeric substance (EPS), biofilm maturation (3D EPS), and dispersion of planktonic cells recolonizing the implant. Surgical site infection (SSI) following breast construction could cause breast implant loss. _S. aureus_ cells could adhere to each other, forming mobile biofilm flocs. Nanotheranostics perform diagnosis and therapy of _S. aureus_. Figure 2: The lifestyle of _Staphylococcus aureus_ biofilms (colonizing the hip implant). The biofilms are multispecies aggregates of cells, secreting a protective extracellular polymeric substance (EPS) contains open fluid channels, resembling a circulatory system. The EPS has heterogeneous compartmentalized microenvironments, resembling tumors, with different oxygen levels (aerobes, fermenters, anaerobes) and pH gradients < 6.5. Bacterial subpopulations of small-colony variants (SCVs, tolerant and resistant to antibiotics) and persisters are formed within biofilms. Persisters are antibiotic tolerant, eliciting recurrent infections, and producing progeny susceptible to antibiotics when resuming growth with no antibiotics. The biofilm EPS and embedded layers of cells constitute a physical barrier, impairing the diffusional penetration of antibiotics and invasion of immune macrophages into biofilms.