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Influence of the synovial environment on infection

Many factors predispose the implant surface to colonization, including the probability that any time an incision is made, bacteria may contaminate it. How then do surgical infection rates remain around 1% for the general population? Nearly, 30 years ago, Gristina (1987) suggested that if the host cells could restore their normal tissue structure on the implant, then bacteria would not be able to colonize the implant surface. Under these conditions, the immune system should be able to clear any contaminants, and no infection would ensue. Thus there would be a “race to the surface” between the cells from the host tissue and the invading pathogens. Gristina hypothesized that if host cell on-growth wins this “race” before infection establishes, then the immune system will clear any invading pathogens and the implant will remain infection free. However, in the joint, this race for the surface may be an unequal race due to the overwhelming effects of synovial fluid on bacteria.

Synovial fluid is a filtrate of serum that is very viscous due to the local production of hyaluronan, lubricin, and other proteoglycans (Gibson and Rooney, 2007; Jebens and Monk-Jones, 1959; Swan et al., 2002). In synovial fluid, bacteria, particularly staphylococci, acquire a biofilm-like, antibiotic-recalcitrant phenotype. Upon initial exposure to synovial fluid, staphylococci form dense aggregates, exhibit slower growth, and become shielded from the effects of antibiotics. These aggregates can be large enough to evade neutrophil capture, may develop into macroscopic clumps, and may be surface-associated or present as floating “biofilms” (Dastgheyb et al., 2015).

The matrix-binding proteins on the surface of staphylococci contribute to the formation of large, fibrous agglomerations of bacteria in synovial fluid. Specifically, key ECM-binding proteins that interact with fibrin (fnbpA, fnbpB, clfA, and clfB) play essential roles in the formation of these macroscopic aggregates. The aggregated phenotype of staphylococci in synovial fluid is the root cause of strong and

Staphylococcal organization in laboratory medium, serum or synovial fluid

Figure 6.1 Staphylococcal organization in laboratory medium, serum or synovial fluid. This figure shows the extent of biofilm formed in trypticase soy broth (TSB), which is a common laboratory medium, in human serum, or in human synovial fluid. In TSB, biofilms are evenly spread, thin, and sparse throughout a sample after a short 24-h incubation time. In serum, biofilms are evenly spread and robust after 24 h of growth. In synovial fluid, biofilms are cloud-like, dense, and aggregated.

rapid biofilm formation. Bacteria incubated in synovial fluid form denser, characteristic biofilms than can be seen when incubated in serum or ideal medium (Fig. 6.1; Dastgheyb et al., 2015).

Importantly, when these dense, proteinaceous aggregates are formed, they are physically inaccessible to antibiotics and induce an altered metabolic state that affects antibiotic effectiveness. Cefazolin, the preoperative antibiotic of choice for orthopedic procedures, is a case in point. This antibiotic has previously been evaluated for bioavailability in synovial fluid and has been found to be present at high levels in synovial aspirates (Dastgheyb et al., 2015; Schurman et al., 1978). Studies have shown that antibiotic penetration in the knee is upwards of 200 gg/mL, and that the antibiotic remains unaltered, and unsequestered by the proteins within synovial fluid (Dastgheyb et al., 2015). Despite this high antibiotic availability, staphylococci have been shown to persist and form biofilms and large aggregates in synovial fluid containing antibiotics (Fig. 6.2). Importantly, the dispersion of these aggregates (enzymatically) in synovial fluid results in an increased antibiotic efficacy. In ideal laboratory media, staphylococcal aggregation decreases responses to cefuroxime, erythromycin, rifampin, vancomycin, and cefazolin (Dastgheyb et al., 2015).

Not surprisingly, considering the effects of synovial fluid and the known properties of biofilm bacteria, infections of joint implants are considered to be relatively indolent. Indolence occurs when bacteria enter a depressed metabolic state that results in decreased production of virulence factors and by implication, decreased damage to the host. This suppressed metabolic state is characterized by decreased proliferation and hence DNA replication and transcription, decreased protein synthesis, and decreased cell wall synthesis, which are all major targets for antibiotic action. Changes in virulence associated with the synovial environment impact the ability of the bacteria either to elicit an acute pyrogenic response or to persist without clear signs of infection (Kim et al., 2015; Monk et al., 2008; Updegrove et al., 2015). The most important virulence factors for pathogens generally contribute to immune evasion, colonization, immune stimulation, or factors that cause direct damage to the host (Massey et al., 2006; Patti et al., 1994; Arciola et al., 2011).

Given the lag time for manifestation of clinical signs of infection, it is important to determine the balance between indolence and virulence of bacterial aggregates in

Staphylococcal aggregate formed in synovial fluid

Figure 6.2 Staphylococcal aggregate formed in synovial fluid. Within 20 min of incubation in synovial fluid, S. aureus aggregates, even in synovial fluid containing antibiotics. Shown is an example of such an aggregate, visualized by scanning electron microscopy. Provided by Rocky Mountain Laboratories, Hamilton, Montana.

synovial fluid. In Staphylococcus aureus, many major secreted virulence factors are controlled by the accessory gene regulator, or agr operon, a global regulator of the staphylococcal virulon. Among these, virulence factors are the phenol-soluble modulins (PSMs), which directly and significantly increase the pathogenicity of S. aureus in in vivo studies (Berube et al., 2014; Cheung et al., 2014) and have been shown to induce neutrophil lysis (Chatterjee et al., 2013). Decreased agr expression has been directly linked to antibiotic recalcitrance and increased aggregation of bacteria (Beenken et al., 2010). In synovial fluid, S. aureus strongly suppresses expression of agr. This suppression results in a >100-fold decrease in the expression of PSMs and delta toxin (Dastgheyb et al., 2015).

After initial aggregation has occurred, synovial fluid has been shown to cause an increase in the expression of MSCRAMMs (matrix surface component recognizing adhesive matrix molecules), such as Fnbp and ClfA, in synovial fluid meaning that biofilm adhesion is increasing, while disaggregation is inhibited. This increased expression leads to the buildup of PIA/PNAG, teichoic acids, and eDNA within the dense matrix, and results in mature biofilms that are recalcitrant to antibiotics (Dastgheyb et al., 2015).

The biofilms found in the synovial joint, whether attached to surfaces or present in the synovial fluid as aggregates, have fibronectin and fibrin as integral components; classical biofilm markers such as polysaccharide intercellular adhesion (PIA) and extracellular DNA are also present (Dastgheyb et al., 2015; Le et al., 2014). Thus the clinically relevant joint biofilm, which is the predominant state in synovial fluid, is a densely packed matrix that is comprised of bacteria adhered to cross-linked proteins encased in the biofilm slime and is therefore robust and recalcitrant to antibiotics. Thus these biofilms cause special problems in the treatment of periprosthetic joint infections.

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