The process of new biofilm formation can be traced back to the adhesion stage of planktonic cells to specific surfaces or tissues. This essential step is under investigation by many researches; several microbial species and their related surface proteins may in fact represent a potential target of anti-adhesion compounds. The main obstacle for the adhesin characterization is the interaction of multiple adhesion receptors with various affinities.

Sortases are membrane enzymes, commonly found in Gram-positive bacteria, that allow the covalent anchoring of surface proteins to peptidoglycan (Marraffini et al. 2006; Mazmanian et al. 1999; Montanaro et al. 2011). This dynamic surface structure permits several interactions between bacteria and their environment and is fundamental to the biofilm development. Projects on the bacteria genome report that sortase is an enzyme with wide substrates. For each analyzed family, 20 potential sortase substrates have been identified in S. aureus, including protein A (Spa), fibronectin-binding proteins (FnbpA, FnbpB), clumping factor (ClfA, ClfB), collagen adhesion protein (CnA), heme-binding proteins (IsdC, IsdB, IsdA), and other surface proteins. These results have been confirmed in other Gram-positive microorganisms, as, for instance, S. epidermidis and S. mutans. Sortase A (srtA) mutants instead did not show all the surface proteins (Marraffini et al. 2006). Paving the way for future research, tests for sepsis, abscess, septic arthritis, and endocarditis on animal models show that the srtA mutant type is less aggressive than wild-type kinds (Marraffini et al. 2006; Jonsson et al. 2003; Weiss et al. 2004; Gianfaldoni et al. 2009). These outcomes have encouraged researchers to think that sortase could work well as a good drug target for anti-adhesion activities. Up to now, researchers have looked for sortase inhibitors through natural and synthetic compounds (Maresso and Schneewind 2008). Aggregates extracted from Cocculus trilobus and Coptis chinensis did manage to stop the attachment of microorganisms to fibronectin (Kim et al. 2002), while synthetic compounds, like (Z)-diarylacrylonitriles, successfully passed the researchers’ screens.

As mentioned earlier, the second stage of biofilm formation is the maturation stage, when cell-to-cell communication is exploited by microorganisms to control the expressions of the genes that are needed and/or that might be directly involved in the development of the biofilm itself. The lack or failure of cell-to-cell communication may make cells preserve their planktonic state.

Quorum sensing (QS) is a specific microbial type of cell-to-cell communication specific for cell density as well as cell population. Bacteria can detect the presence of other microorganisms nearby by using small molecules secreted by these microorganisms themselves. This observation reveals that bacteria don’t act as independent individuals; instead, they behave as a population. In light of this coordination, interferences with the quorum sensing might thus impede bacteria’s colonization of an implant (Njoroge and Sperandio 2009; Kaufmann et al. 2008). QS was first described in the 1960s by Tomasz (1965); since then, research efforts have identified and described alternative types of QS, among which are N-acyl homoserine lactone (AHL) system and 4-quinolone system from Gram-negative bacteria, AgrD peptide systems for Gram-positive bacteria, AI2/LuxS systems for both Gram-negative and Gram-positive bacteria, and farnesol systems for fungi.

This identification process has come up with many different quorum-sensing inhibitors (QSI); there are two classes that could be potential leading candidates: the furanones and RNAIII-inhibiting peptide (RIP) ones. There are some superior and inferior plants that are producing QSI, as the algae Delisea pulchra, which is an endemic type of algae from Southeastern Australia, producing halogenated furanones that impede the bacterial colonization and the formation of biofilm (Manefield et al. 1999). Furanones prevent the bacteria’s settlement and the formation of biofilm by interfering with a fundamental bacteria QS pathway, the AHSL regulatory system in Gram-negative microorganisms. Furanones also interfere with the AI-2 signaling system, generally related to both Gram-negative and Gram-positive bacteria. So far, synthetic programs have proposed a library consisting of up to 200 furanones and furanone analogues, including surface-attached furanone, with its strong in vitro and in vivo antipathogenic characteristics (Lazar 2011). When tested on models of animal disease, furanones did help the host immune system to effectively tackle infections (Wu et al. 2004; Hentzer et al. 2003), and they also did control S. epidermidis biofilm formation in vitro, as well as in a sheep model when covalently bound to catheters (Hume et al. 2004). Preliminary results are appealing, but furanones have not been applied to animal models of orthopedic implant-related infections yet.

In vitro applications also identified N-acyl homoserine lactone-based QS system as a predictable target preventing Pseudomonas biofilm formation (Geske et al. 2005; Kaufmann et al. 2006). Animal models with polymeric joint-related infections also proved the effectiveness of QS inhibitors in augmenting the vulnerability of bacterial biofilms when subjected to standard antimicrobial agents, such as antibiotics and detergents (Musk and Hergenrother 2006; Pan and Ren 2009; Brackman et al. 2011). Christensen et al. (2012) recently reported intraperitoneal or subcutaneous injection of the QS inhibitor furanone C-30, ajoene, or horseradish juice extract in combination with tobramycin in a mice model of infection. In this model, they inserted silicone tube implants, previously colonized with wild-type P. aeruginosa, into the mice’s peritoneal cavity. For each QS inhibitor tested, the treated group experienced a significant reduction in the formation of colony compared to placebo.

On the other side, the RNAIII-inhibiting peptide (RIP), in amide form, has effectively inhibited virulence, the onset of biofilm, as well as antibiotic resistance of staphylococci. Kiran et al. (2008) not only proved the effectiveness of RIP against some strains of methicillin-resistant S. aureus (MRSA); they also found another RIP, hamamelitannin, which is analogue to the nonpeptide RIP and can impede implant-related MRSA infections in a concentration-dependent manner. This proved to be successful on some animal models, but in vitro studies revealed that the stability and toxicity of the product could be of major concern (Balaban et al. 1998; Giacometti et al. 2003; Anguita-Alonso et al. 1998; Lovetri and Madhyastha 2010; Cirioni et al. 2007).

NSAIDs are cyclooxygenase inhibitors able to significantly slow down the in vitro formation of biofilm both in fungi and in bacteria. This cyclooxygenase-dependent synthesis of fungal and bacterial prostaglandin(s) might in fact be relevant in terms of biofilm formation and morphogenesis, and, last but not least, it might also contribute to the regulation phase of these two physiological processes. Approximately 10 years ago, the study carried out by Faber and coworkers revealed that NSAIDs were able to impede the attachment of Staphylococcus epidermidis to medical polymers (Farber and Wolff 1992). An analogous effect has been confirmed in more recent studies on aspirin, etodolac, and diclofenac: among these, aspirin has even implied a 95 % inhibition of Candida albicans biofilm development. Celecoxib, nimesulide, ibuprofen, and meloxicam did prove able to impede biofilm formation, but with a less dramatic effect than aspirin. Aspirin also turned out to be effective in the treatment of already formed and growing biofilms. To this extent, the efficacy of aspirin was related to the dose applied, and it proved its inhibitor property at pharmacological concentration. Still, this inhibition effect was canceled out when prostaglandin E2 was simultaneously added. At 1 mM, aspirin reduced the viability of biofilm organisms to 1.9 % of that of controls (Alem and Douglas 2004). Also ibuprofen displayed some inhibition properties when tested on the S. pneumoniae and E. coli strain biofilm formation (del Prado et al. 2010; Naves et al. 2010); some effects also emerged with respect to the attachment of Escherichia coli to epithelial cells (Drago et al. 2002). Despite these promising results, these inhibition effects emerged only in in vitro studies: further research on animal models is thus needed to confirm the predicted therapeutic efficacy of NSAIDs on biofilm formation on implanted biomaterials.

Antimicrobial peptides are widely diffused molecules in living organisms, whose extensive host defense property makes them fundamental as part of innate immunity. These large molecules, with a net positive charge, contain approximately 50 % hydrophobic residues. Their working mechanism binds the structural molecules that are negatively charged to the microbial membrane. AMPs target a wide range of microbial activity, rarely able to resist them, and they have been described as able to prevent the onset of biofilm on stainless steel surface. Héquet et al. (2011) proved in fact that antibacterial peptides magainin I and misin, with their covalent bound to stainless steel surfaces, were able to reduce in a significant way the attachment of Gram-positive Listeria ivanovii. Melamine, a nonhemolytic hybrid peptide between melittin and protamine, decreased the adhesion of bacteria to the contact lenses, to which it was covalently bound, and did not lead to any resistance against S. aureus or P. aeruginosa during the repeated passage in subminimal inhibitory concentrations (Willcox et al. 2008). Moreover, P. aeruginosa guinea pig models revealed that silicone hydrogel lenses with melamine led to a reduction in contact lens-induced acute red eye, and S. aureus rabbit models suggested that they also hindered contact lens-induced peripheral ulcers. Citropin 1.1, extracted from Litoria citropa, the green tree frog, is powerful to the detriment of biofilm activity and is even more powerful against S. aureus biofilm when provided together with rifampin and minocycline (Cirioni et al. 2006). A relevant reduction in the bacterial counts of biofilm emerged in a model of S. aureus infection, where rats were treated with central venous catheters previously treated with citropin 1.1 peptides and/or antibiotics (Høiby et al. 2010). The working principle of AMPs include membrane-disrupting action, functional inhibition of proteins, binding with DNA, and detoxification of polysaccharides (lipopolysaccharide and lipoteichoic acid) (Park et al. 2011). The pitfalls related to AMPs treatments are many, as their large-scale production is costly and complicated. Moreover, they are also sensitive to protease digestion. Modified versions of AMPs led to the formation of synthetic antimicrobial peptides, known as SAMPs (second generation of AMPs). Their working mechanism is similar to the AMPs’, but SAMPs have better pharmacokinetic properties. This second generation is thus an improvement over the former version, but they are still broad spectrum. As a response, recent studies came up with STAMPs, that is, selectively targeted antimicrobial peptides, to target species-specific biofilm control. In other words, STAMPs are the third generation of AMPs, as they are their modified recombinant version (Eckert et al. 2006; He et al. 2009). The in vivo activity of these compounds has still to be evaluated (Li et al. 2010).

To tackle staphylococcal biofilm disruption, many different agents have been considered. Among them, the encouraging results are related to those enzymes capable of attacking biofilm components. Initially, Selan and her coworkers recommended the use of proteolytic enzyme serratiopeptidase for the treatment of biofilm-related infections (Selan et al. 1993; Artini et al. 2011b). Almost 20 years later, in a rat model of joint-related infection due to S. epidermidis strain, with a high slime production capacity, serratiopeptidase was injected in the knee affected: the subsequent antibiotic therapy reported a 94.4 % infection healing rate, higher than the 62.5 % cure rate emerged from groups subjected to the antibiotic treatment only. The resulting difference was confirmed to be statistically significant (Mecikoglu et al. 2006). In addition to this enzyme, research has also investigated the biofilm-dispersing property of deoxyribonuclease I (DNase I) and dispersin B (DspB). Whereas the former degrades extracellular DNA, which is a new structural component contributing to the biofilm steadiness, the latter hydrolyzes the biofilm poly-(beta-1,6)-N-acetylglucosamine exopolysaccharide, also called polysaccharide intercellular adhesin. DNase I also inhibits the formation of biofilm, when present in the culture medium, right at the stage of bacteria onset, but DNase I fails to separate biofilm once it has already formed. More specifically, DNase I is mainly associated to an increase in the biofilm bacteria sensibility to killing by several biocides and to the detachment by anionic detergents and not to the inhibition or detachment of biofilm formation or already formed biofilm. Thus, following Kaplan (2009), biofilm detachment might occur rapidly with DNase I and at a clinically achievable concentration of the enzyme (Kaplan et al. 2012).

Darouiche et al. (2008, 2009) discovered that DspB, combined with antiseptics (triclosan or chlorhexidine), boasted the broad-spectrum antibiofilm and antimicrobial activity against S. aureus, S. epidermidis, and Escherichia coli to coated vascular catheters; moreover, catheters coated with triclosan and DspB effectively showed an in vivo antibacterial activity by subcutaneous implantation of segments in a model with S. aureus-infected rabbits.

An ancient approach that considered maggot or larvae therapy can be brushed up and applied in nowadays context: to this purpose, a living and disinfected fly larva is introduced into a nonhealing skin or into soft tissue wounds of animals or humans. This cleans out the necrotic tissues and disinfects and debrides the wound. The Lucilia sericata excretions and secretions inhibited the biofilm formation up to 92 %, and this effect has been confirmed by several in vitro applications on various microorganisms (Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis, and Enterobacter cloacae) and on several biomaterials, such as polyethylene, titanium, and stainless steel, all currently used in orthopedic practices (Harris et al. 2009; Cazander et al. 2010).

NAC is a mucolytic agent with antibacterial properties. This molecule is an antioxidant containing thiol and breaks disulfide chemical ties in mucus (Sheffner 1963) and competitively impedes amino acid (cysteine) use (Zygmunt and Martin 1968). NAC also diminishes the attachment of staphylococci, as well as the adhesion of some Gram-negative bacilli, to some abiotic materials (Perez-Giraldo et al. 1997; Olofsson et al. 2003; Mansouri and Darouiche 2007; Schwandt et al. 2004). In addition to this, NAC slows down the formation on an extracellular polysaccharide matrix, and, in the meanwhile, it enhances the rupture of mature biofilms. According to our data (Drago et al. 2013), NAC, when dissolved at a final concentration of 100 mg/ml, successfully disrupted S. aureus– and P. aeruginosa-related biofilm, on disks of polyethylene and titanium, already 3 h after incubation (Fig. 22.2). Still, some differences in the biofilm eradication emerged when targeting already formed P. aeruginosa biofilm, according to the type of disk tested: polyethylene displayed a 50 % rate, whereas eradication was successful only on 20 out of 100 titanium disks. The disaggregation of biofilm was monitored for the following 18 h. Figure 22.2 displays the confocal laser scanning microscopy analysis of biofilms untreated and treated with NAC, 18 h after incubation. Live cells, with their intact cell membranes, were stained with Syto9 and were identifiable by their green fluorescence. Furthermore, SYPRO^® Ruby stain identifies the extracellular polysaccharide matrix and comes with a red fluorescence. Untreated biofilms were mainly red, but they also contained a green cell that revealed the presence of bacteria in the extracellular polysaccharide matrix. When treated with NAC, after 18 h from incubation, the same analysis revealed a reversed scenario, with more greens and less reds, thus suggesting the successful disruption of biofilm bacteria carried out by NAC agents. Disrupted bacteria returned to their planktonic phenotype. NAC also displays some bactericidal properties, against bacteria clinically relevant and resistant to drugs. Recent studies have tested NAC on organism viability during the planktonic and biofilm stages; the microorganisms tested were, for instance, the methicillin-sensitive and methicillin-resistant S. aureus and S. epidermidis, the vancomycin-resistant Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumonia, Candida albicans, and C. krusei. At the 80 mg/ml concentration, NAC proved to be bactericidal (with a more than 99.9 % reduction) with respect to all the tested bacteria. On top of that, there were no recoverable organisms only 30 min after incubation and NAC proved to be fungistatic against Candida species. In the interval 5–10 mg/ml, NAC displayed the lowest inhibition and bactericidal concentration. The thickness of biofilms treated with NAC decreased significantly on all organisms but vancomycin-resistant enterococci (Aslam and Darouiche 2011).

Agents disrupting biofilms may be exploited in a number of ways, but the pretreatment aimed at the removal of biofilm residues deserves further attention. An analysis carried out by the water industry has revealed that different surfaces homogenously attract planktonic cells, and, when contaminated by organic materials, especially residual biofilm matrices, this attraction process occurs at a speed at least ten times higher. Biofilm formation can occur already at the orthopedic implant manufacturing stage, more specifically when it involves machining techniques using a wet interface between the tool assembly and the implant. To this purpose, sterilization, for instance, ethylene oxide, kills the bacteria on the biofilm but is not enough to totally remove the residues from their matrices. These should be eliminated, applying the available enzyme treatments, before the joint implantation (Olofsson et al. 2003).

When combined with various antibiotics, some synergies emerge. For instance, the combination of NAC and fosfomycin and tigecycline is able to reduce S. aureus, S. epidermidis, as well as some Gram-negative bacteria (Marchese et al. 2003; Aslam et al. 2007). El-Feky et al. (2009) obtained analogous results in their analysis of nosocomial staphylococci and Gram-negative bacilli retrieved from removed ureteral stents, using NAC (2 and 4 mg/ml) with ciprofloxacin. When comparing the treated and control groups, they obtained is a 94–100 % biofilm formation inhibition rate and a 86–100 % disruption of preformed biofilms. Zhao and Liu (2010) proved that the minimum inhibitory concentrations (MICs) of NAC for most P. aeruginosa were achieved at 10–40 mg/ml, whereas at a 0.5 mg/ml concentration NAC would be able to disrupt already formed P. aeruginosa biofilms. In their study, the level of biofilm disruption was proportional to the level of NAC concentration, and total disruption was achieved at the 10 mg/ml concentration. Already at 2.5 mg/ml and at >2 MIC, NAC and ciprofloxacin respectively displayed successful P. aeruginosa biofilm killing properties. Different combinations of NAC and ciprofloxacin reduced, with respect to the control group, viable biofilm-related bacteria. The most synergic result was obtained at a 0.5 mg/ml NAC concentration, combined with a ½ MIC of CIP. Moreover, the production of extracellular P. aeruginosa polysaccharides dropped by 27.64 and 44.59 % at 0.5 and 1 mg/ml NAC concentration, respectively. The studies so far mentioned all report in vitro results, and, over time, clinical experience has made big improvements in the treatment of respiratory tract infections. All these elements make NAC as an ideal antibiofilm agent when dealing with implant-related infections. Still, further animal models are needed to confirm its successful application.