To understand the diagnostic principles and treatment models of PJI, it is imperative to understand the development and functions of biofilm. The transition from planktonic growth to biofilm occurs in response to environmental changes, such as the presence of a prosthetic device, and involves multiple regulatory networks. This communication termed “quorum sensing” (QS) is analogous to the paracrine signaling in multicellular organisms and enables the bacteria to regulate their gene synthesis (Gristina and Costerton 2009).

At the end, biofilm formation enables single-cell organisms to assume a temporary multicellular lifestyle, in which “group behavior” facilitates survival in adverse environments. What was once defined as the formation of a community of microorganisms attached to a surface has come to be recognized as a complex developmental process that is multifaceted and dynamic in nature (Kostakioti et al. 2013).

This cellular reprogramming alters the expression of surface molecules, nutrient utilization, and virulence factors and equips bacteria with an arsenal of properties that enable their survival in unfavorable conditions (Whiteley et al. 2001; Stanley et al. 2003; Vuong et al. 2004; Lenz et al. 2008).

As soon as a new device gets implanted in a surgical site, a process notoriously called “race for the surface” begins (Gristina et al. 1995).

According to this concept, there is a competition for colonizing the surface between extracellular matrix proteins (fibrinogen, fibronectin, vitronectin, thrombospondin, bone sialoprotein) and eukaryotic cells (fibroblasts, osteoblasts, endothelial cells) on one hand and prokaryotic bacterial cells on the other. Matrix proteins are known to cover foreign material as soon as it appears in the human body. In the next step, fibroblasts interact with a layer of matrix proteins using specific receptors called integrins. In such a way, the implant becomes covered by a viable barrier, capable of defense functions against bacteria. In case there are bacteria present at the time of implantation, they enter the competition for the surface, and the outcome largely depends on their number and features.

Bacterial adhesion to biomaterial surfaces and development of biofilm are thought to consist of four separate stages regulated through different mechanisms among which the best studied is QS (Lazar 2011; Costerton et al. 2007) (Fig. 21.2).

Stage I can be subdivided in to two phases. In the first, bacteria inoculated in planktonic form diffuse through fluids propelled by nonspecific physical factors like gravity, Brownian motion, surface tension, and van der Waals bonds. While this process for the majority of pathogen is at least in part stochastic, motile bacteria such as Pseudomonas aeruginosa have a competitive advantage. When bacteria come into proximity of an implant at distances smaller than 3 nm, they start interacting with biomaterial by means of hydrogen, ionic, and hydrophobic bonds. Energy needed for disruption of these bonds is small; therefore, initial attachment is dynamic and reversible (Donlan 2002; Krekeler et al. 1989; Kostakioti et al. 2013).

The second phase, mediated by multiple macromolecules able to bond with the implant surface and in general called adhesins, is less reversible. Microorganisms appear to have different adhesins for different surface materials (Hasty et al. 1992).

Pseudomonas aeruginosa, Escherichia coli, and others, mainly Gram-negative bacteria, connect to implant surface using flagella (Darouiche 2001). Flagella are protein hooks sized 20 nm used for grabbing the surroundings (An and Friedman 1998). Some bacterial species adhere to implant surface using specific means, e.g., Staphylococcus aureus uses MSCRAMM adhesive molecule (microbial surface components recognizing adhesive matrix molecules) to connect with extracellular matrix proteins covering the implant (Patti et al. 1994). Adhesion to a surface leads to important changes in many aspects of bacterial metabolism. Genes needed for biofilm development become activated. There are also other prerequisites for induction of this process like availability of nutrients, temperature, pH, osmolality, availability of iron, and presence of different signal molecules from other bacteria. During this stage, bacterial cells form a monolayer and exhibit a logarithmic grow rate.

Stage II, also called accumulation phase, is characterized by the irreversible binding to the surface and begins from minutes to hours after the first stage. Once adherence has taken place, the now sessile (or fixed) organisms begin to multiply and forming microcolonies while emitting chemical signals that “intercommunicate” between bacterial cells (Høiby et al. 2010a).

When the signal intensity exceeds a certain threshold level, the genetic mechanisms underlying extracellular polymeric substances (EPSs) production are activated. EPSs consist primarily of polysaccharides and contribute 50–90 % of the organic matter in biofilms. This matrix is highly hydrated (98 % water), and apart from water and microbial cells, it is a very complex material. Biofilm in fact is not only tenaciously bounded to the underlying surface but also has a propensity to act almost as filters to entrap particles of various kinds, including minerals and host components such as fibrin, RBCs, platelets, and planktonic bacteria (Costerton et al. 1999).

Therefore, all major classes of macromolecules (proteins, polysaccharides, and nucleic acids) are present in addition to peptidoglycan, lipids, phospholipids, and other cell components (Taraszkiewicz et al. 2013).

In case of Staphylococcal infection, this second stage is mediated by multiple molecules including polysaccharide intercellular adhesin (PIA)/poly-N-acetyl glucosamine (PNAG); proteins such as biofilm-associated protein (Bap), accumulation-associated protein (Aap), or fibronectin-binding protein (FnBp); and eDNA (Rohde et al. 2010).

Stage III is also known as maturation; it is characterized by the synthesis of tower-like structures containing, in part, eDNA from lysed bacteria and the generation of multiple metabolic niches that include bacteria growing under aerobic conditions, microaerobic-anaerobic conditions, dormant, and dead cells.

The production of QS signals bacterial surface components such as exopolysaccharides (EPSs) is required for the formation of a mature biofilm.

In this stage biofilms reach their ultimate thickness, generally greater than 100 μm with mushroom-shaped or tower-like microcolonies (Zoubos et al. 2012).

At this stage the biofilm shows maximum resistance to antibiotics (Høiby et al. 2010b).

Stage IV, during this stage, maybe due to bacteriophage activity within the biofilm, focal areas of the biofilm dissolve, so that some of the bacteria develop the planktonic phenotype and leave the biofilm, such event may cause metastatic expansion of the infection and a reactivation of clinical features (Webb et al. 2003; Gristina et al. 1987) (Figs. 21.3 and 21.4).

Using QS, biofilm bacteria behave very intelligently, adjusting the growth of colonies in response to various challenges; the complexity of biofilm structure and metabolism has led to the analogy of biofilms to tissues of more complex organisms (Costerton et al. 1995).

Living in biofilms, the bacteria are protected from deleterious conditions; this structure and its physiological attributes confer an inherent resistance to antimicrobial agents, whether these antimicrobial agents are antibiotics, disinfectants, or germicides. Cells in different regions of a biofilm exhibit different patterns of gene expression; likewise, growth, protein synthesis, and metabolic activity are stratified in biofilms (Davies et al. 1993).

Even if the structures that form biofilms contain channels in which nutrients can circulate (An YH and Friedmann RJ 1998), inspection of environmental as well as in vitro biofilms has revealed that the oxygen as well as nutrition concentration may be different in different areas of the matrix (Beer et al. 1994; Costerton et al. 1995).

Because of these nutritional and oxygen gradient, bacteria within a biofilm exist in different metabolic zones (Stewart and Franklin 2008). Nutrient-depleted zones can result in a stationary phase – like dormancy within the biofilm – which may be responsible for the general resistance of biofilms forming bacteria to antibiotics. Therefore, limited penetration of nutrients, rather than restricted antibiotic diffusion, may contribute to a generalized resistance or tolerance to antibiotics (Stoodley and Stoodley 2009).

Example of such metabolic heterogeneity is as described by Rani et al. (Rani et al. 2007) that S. epidermidis growing in a biofilm existed in four metabolic states: aerobic, fermentative growth, dead, and dormant.

Due to increased production of endogenous reactive oxygen species and a deficient antioxidant system, the mutation frequency of biofilm-growing bacteria is significantly increased compared with planktonically growing isogenic bacteria, and there is increased horizontal gene transmission in biofilms. Plasmid interchange is largely facilitated inside biofilm. The reason is the close proximity of bacteria; reduction of shear forces by the slime also eases conjugating process (Ehlers and Bouwer 1999).

These physiological conditions may explain why biofilm-growing bacteria easily become multidrug resistant by means of traditional resistance mechanisms against β-lactam antibiotics, aminoglycosides, and fluoroquinolones, which are detected by routine susceptibility testing in the clinical microbiology laboratory where planktonic bacterial growth is investigated. Thus, bacterial cells in biofilms may simultaneously produce enzymes that degrade antibiotics, have antibiotic targets of low affinity, and overexpress efflux pumps that have a broad range of substrates (Driffield et al. 2008; Molin and Tolker-Nielsen 2003).

The matrix can provide bacteria with protection also from host defense mechanisms. Bacterial cells inside the biofilm are well protected from complement system, neutrophilic granulocytes, killer cells, antibiotic peptides, antibodies, and phagocytosis. This impairment of host defense mechanisms has been demonstrated in a number of in vitro models.

Staphylococcus epidermidis is the paradigmatic example of virulence due to biofilm. Such a bacterium is generally not considered pathogenic, and its presence on normal skin and mucosal surface is not normally associated with any signs of inflammatory or immune reactions. However, the host-staphylococcal balance becomes disrupted if the bacterium gains entry into the tissues and especially if it reaches an orthopedic implant. After adhering to the implant surface, Staphylococcus epidermidis secretes a layer of slime that can inhibit the phagocytic activity of neutrophils (Shiau and Wu 1998) and decreases its antibiotic susceptibility significantly making this infection very hard to treat (Sousa 2011; Shiau and Wu 1998).

As Roman’s said “divide et impera,” the better way to fight against biofilm forming bacteria is before the bacterial adherence to the foreign material has taken place, therefore before bacteria assume multicellular lifestyle. In such a “war,” the host, the wound, and all the environmental factors are involved. In the next pages, we will focus on antimicrobial prophylaxis, the role of the antimicrobial therapy in the treatment of PJI, and perspectives for the future possible new molecules and target.