**5.1 Genomic features of staphylococci associated with bone infections**

A number of studies have attempted to identify an association between the possession of certain virulence genes by Staphylococci and invasive disease. Thus Peacock et al.[36]suggested that the possession of certain combinations of virulence factor genes is associated with invasive disease, and increased severity of infection following examination of a panel of 334 S. aureus isolates by PCR. The isolates comprised those from 179 healthy patients, 94 hospitalacquired isolates and 61 community-acquired isolates. Seven putative virulence genes, including the adhesin genes fnbA and cna, the toxin genes sej, eta and hlg, and icaA, which is involved in biofilm production, were found to be associated with invasive isolates. The association with specific types of invasive infection was not examined and indeed the small number of isolates examined in this study would have precluded such an analysis.

The genes for the fibronectin-binding proteins fnbA and fnbB have been reported to be present in 98% and 99% of clinical isolates, respectively, from a range of orthopaedic associated infections, whereas the cna gene, encoding the collagen -binding protein was identified in just 46% of isolates[37]. Another study by Peacock et al.[38]found the

and fungi cause the remainder of cases[30,31]. Moreover, Staphylococcus aureus has the dominance in acute septic arthritis, and is particularly common in patients with rheumatoid arthritis[32]while coagulase -negative staphylococci can be found mainly in periprosthetic

 Other gram-positive bacteria as causative agents for hip joint infections are streptococci, especially Streptococcus pyogenes, Enterococcus faecalis and Corynebacteria species. Propionibacterium acnes is a common cause of infection associated with shoulder

A large number of different gram-negative rods act as infectious agents on joints. The group of enterobacteria contains a broad spectrum of pathogens. Salmonella enterica, Shigella species, and Yersinia species are classically described as pathogens for purulent and reactive forms of arthritis. Pseudomonas aeruginosa can be found more often in predisposed patients (e.g. diabetics). In otherwise healthy people it is associated with iatrogenic modes of

Campylobacter species, however, are classic agents of reactive arthritis, as well as the obligate intracellular bacteria Chlamydia trachomatis, Mycoplasma pneumoniae, and Ureaplasma urealyticum. From the spirochaetales only Borrelia burgdorferi sensu lato is relevant. Less commonly identified organisms for joint infections often accompanied with

Up to 20% of cases are polymicrobial, most commonly involving methicillin -resistant S. aureus (MRSA) or anaerobes, such as Bacteroides fragilis[34]. Approximately 7% of cases are

Infection with virulent organisms (e.g., S. aureus and gram-negative bacilli) inoculated at implantation is typically manifested as acute infection in the first 3 months (or, with hematogenous seeding of the implant, at any time) after surgery, whereas infection with less virulent organisms (e.g., coagulase-negative staphylococci and P. acnes) is more often

A number of studies have attempted to identify an association between the possession of certain virulence genes by Staphylococci and invasive disease. Thus Peacock et al.[36]suggested that the possession of certain combinations of virulence factor genes is associated with invasive disease, and increased severity of infection following examination of a panel of 334 S. aureus isolates by PCR. The isolates comprised those from 179 healthy patients, 94 hospitalacquired isolates and 61 community-acquired isolates. Seven putative virulence genes, including the adhesin genes fnbA and cna, the toxin genes sej, eta and hlg, and icaA, which is involved in biofilm production, were found to be associated with invasive isolates. The association with specific types of invasive infection was not examined and indeed the small number of isolates examined in this study would have precluded such

The genes for the fibronectin-binding proteins fnbA and fnbB have been reported to be present in 98% and 99% of clinical isolates, respectively, from a range of orthopaedic associated infections, whereas the cna gene, encoding the collagen -binding protein was identified in just 46% of isolates[37]. Another study by Peacock et al.[38]found the

osteitis or osteomyelitis are Brucella species and Mycobacterium tuberculosis.

culture-negative, often in the context of previous antimicrobial therapy[35].

manifested as chronic infection several months (or years) postoperatively.

**5.1 Genomic features of staphylococci associated with bone infections** 

infections and after diagnostic arthroscopies.

infection during diagnostic procedures.

An overview is shown in Table 2[8].

**5. Interaction of staphylococci with bone** 

arthroplasty[33].

an analysis.

prevalence of both fnbA and fnbB genes, as opposed to just one of the two, to be significantly higher in invasive isolates than in 'carriage' strains in a panel of 163 strains, which included septic arthritis and osteomyelitis isolates. Genes encoding Panton-Valentine leukocidin were found to be present in 59 of 89 S. aureus isolates from cases of acute haematogenous osteomyelitis. The presence of pvl genes is associated with an increased risk of severe infection requiring intensive care, bacteremia and more severe systemic inflammation [39,40]. However, one of the problems with the above studies is that it is unclear how representative these strain collections are of those isolates carried in other establishments and regions across the world, since strain typing was not reported.

Strain typing studies of S. aureus, using multilocus sequence typing (MLST) and comparative genomic microarray hybridizations have so far failed to identify any specific clonal lineages associated with invasive disease. However, these studies did not use a collection of isolates from specific invasive diseases and therefore do not rule out the possibility that specific lineages or genes are associated with specific types of infection, such as osteomyelitis or septic arthritis.

To date, the only genome comparison study relevant to S. aureus bone infections has been done using comparative genome microarray hybridisations of the S. aureus UAMS-1 strain, isolated from an osteomyelitis patient, with a range of genome sequenced strains[41]. These authors found variations in the complement of adhesin, toxin, exoenzyme and regulatory genes. Although it is not possible to draw general conclusions about association with bone infection from characterisation of a single strain, the presence of fnbA, but not fnbB or the bone sialoprotein-binding gene bbp, in UAMS-1 suggest that fnbB and bbp are dispensable for bone infection, at least in certain genetic backgrounds. Thus at this juncture there is a lack of evidence to support or disprove an association between specific S. aureus lineages or specific genomic features and the pathogenesis of bone infections.

### **5.2 Bone as a target organ**

In terrestrial vertebrates mature bone is made up of dense surface plates of bone, known as the cortices, and within these is a network of bone struts oriented to oppose loading forces, known as trabecular bone. Trabecular bone is typically replaced every 3–4 years, with the denser cortical bone taking over a decade to replace in adults. This process of continual remodelling is required to remove old bone and microfractures to ensure bone integrity and mineral homeostasis. The skeleton is a dynamic organ system, in a state of perpetual turnover which is continually remodelled by the actions of two cell types, osteoblasts and osteoclasts.

Osteoblasts are responsible for the deposition of bone matrix; they are found on bone surfaces and are derived from mesenchymal steoprogenitor cells. These cells secrete osteoid, a mixture of bone matrix proteins primarily made up of type I collagen (over 90%), proteoglycans such as decorin and biglycan, glycoproteins such as fibronectin, osteonectin and tenascin-C, osteopontin, osteocalcin and bone sialoprotein, oriented along stress lines. Osteoblasts are also thought to facilitate the mineralization of bone matrix, whereby hydroxyapatite, [Ca3(PO4)2]3·Ca[OH]2, crystals form, making up around 90% of bone matrix. It is thought that 'nucleators' are required to instigate mineralisation, and phosphate-containing matrix proteins like bone sialoprotein and osteopontin are likely to play such a role. Osteoblasts also produce tissue nonspecific alkaline phosphatase (TNAP) which cleaves phosphate esters to liberate free inorganic phosphate, which is key to the process of mineralisation[42].

Staphylococcus Infection Associated with Arthroplasty 467

1(activator protein 1) and NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin dependent 1) which drive osteoclast differentiation[44]. Osteoprotegrin (OPG) is an endogenous inhibitor of RANKL signalling, functioning as a decoy receptor that binds to

A number of host cytokines play a significant role in the pathogenesis of osteomyelitis, and there is strong evidence that production of these cytokines is induced by staphylococcal infection of bone, and that they directly contribute to bone destruction. In particular, the inflammatory cytokines tumour necrosis factor α (TNFα), interleukin 1 (IL-1) and IL-6 seem to be especially important in bone physiology and pathology. In patients with acute osteomyelitis, plasma levels of TNFα, IL-1β (the secreted form of IL-1) and IL-6 are all elevated. High levels of IL-1β, IL-6 and TNFα are also found in the synovial fluid of patients with septic arthritis. Interestingly, specific polymorphisms in the IL-1α and IL-6 genes have recently been found to be associated with an increased risk of osteomyelitis in the Greek population[49]. The most recently identified member of the IL-1 cytokine superfamily is IL-33, has been shown to expressed in differentiated osteoblasts and blocks osteoclast

A number of animal models of *S. aureus* osteomyelitis reveal that bone infection can lead to elevated levels of these cytokines both locally and systemically. Increased levels of IL-1β have been measured in the tibiae of 22-month-old rats experimentally implanted with *S. aureus* infected needles, and the same animals have increased circulating levels of IL-6[51]. In a murine osteomyelitis model, bone levels of IL-1β and IL-6 are significantly increased in the early post-infection period, with TNFα rising later during the infection. The local source of these cytokines is not fully clear. Production of IL-1β can be induced in human osteoblastlike cell lines by a variety of stimuli, including TNFα. However, infection of primary mouse osteoblasts with *S.aureus* results in increased transcription, but not increased protein synthesis or secretion of IL-1β. TNFα is detectable only at low levels in human osteoblasts derived from mesenchymal stem cells and the osteosarcoma cell line MG63.Infiltrating immune cells may therefore be a more likely source of IL-1β and TNFα in bone in response to infection. IL-6 however, is produced by osteoblasts in response to a variety of signals,

These cytokines have potent effects on the process of bone remodelling, and are strongly implicated in the pathology of osteomyelitis. Cell culture models support the view that IL-1 and TNFα stimulate the proliferation and differentiation of osteoclast progenitors into mature osteoclasts in the presence of osteoblasts. TNFα and IL-1β also stimulate osteoclastmediated bone resorption, a process which may also require the presence of osteoblasts. Similarly, IL-6 increases bone resorption activity and osteoclast number in cultured mouse calvariae, and stimulates osteoclast differentiation in the presence of osteoblasts. In vivo, local administration of IL-1 and TNF antagonists in a non-human primate model of periodontitis results in significant reduction of osteoclast formation and bone destruction. Intravenous administration of TNFα and IL-1 in mice stimulates bone resorption in a dosedependent fashion, and deletion of the murine IL-1R, TNF-R1 and TNF-R2 receptors and of caspase-1 significantly decreases osteoclast number and the area of bone resorption in

IL-1β and TNFα also inhibit the differentiation of mesenchymal stem cells into osteoblastlike cells, and suppress the accompanying mineralisation and increased expression of

RANKL and prevents its association with RANK.

formation from bone marrow precursor cells[50].

including infection with *S. aureus* .

calvariae following lipopolysaccharide (LPS) injection.

**5.3 Inflammation in bone infection** 

Osteoblasts are not terminally differentiated, and some may form osteocytes and become implanted in the bone matrix, eventually ceasing the secretion of osteoid, whilst others undergo apoptosis. Osteocytes are also involved in bone maintenance, detecting stress within the bone through echanosensitive mechanisms located in extensive cellular projections, called canaliculi, that interconnect osteocytes[42]. Osteocytes are thought to respond to mechanical stress by undergoing apoptosis, leading to osteoclast recruitment and differentiation, possibly by alterations in the levels of soluble factors produced by the osteocyte. Candidates include transforming growth factor β(TGF-β), which may suppress osteoclastogenesis when produced by healthy osteocytes[43,44].

Osteoclasts, which are multinucleate cells derived from the acrophage-monocyte lineage, perform the opposing action of bone matrix removal. These cells express large quantities of a vacuolar-type H+-ATPase on their cell surface, along with chloride channel 7 (ClC 7)enabling localised hydrochloric acid secretion into a closed compartment, known as the resorption lacuna, and subsequent olubilisation of bone mineral. The cell is attached to the bone matrix by a sealing zone membrane to create this compartment, and fusion of acidified vesicles with the plasma membrane contributes further to acid release[45].Following mineral solubilisation, proteolysis of bone matrix proteins is then possible. Cathepsin K is centrally involved in degradation of bone matrix, it is highly expressed by osteoclasts and digests substrates such as collagen and osteonectin. Evidence from knock-out mouse and selective inhibitor experiments indicates that cathepsin L, and MMPs also play a role in degrading bone matrix[46]. Osteoclasts also secrete acid phosphatases, such as tartrate-resistant acid phosphatase(TRAcP), which is used as an osteoclast marker and is activated by cathepsin K cleavage. TRAcP is able to generate reactive oxygen species in addition to having phosphatase activity. The exact cellular function of TRAcP in bone resorption is not well understood, but serum TRAcP levels correlate with bone-resorptive activity, and TRAcPdeficient mice exhibit reduced osteoclastic bone resorption and increased bone mineralization.

The balance of activity between these two cell types is crucial to maintaining the proper homeostasis of bone turnover, and any shift in the relative levels of osteoblast and osteoclast activity can result in bone pathology. Infection with a pathogen such as S. aureus is capable of stimulating such a shift, mediated in part by induction of an inflammatory response. There is intimate interaction between the two cell types, with osteoblasts interpreting the majority of extracellular signals and subsequently modulating osteoclast differentiation and function[44].

Interaction between the RANK (receptor activator for nuclear factor κB) receptor, expressed by osteoclast precursors, and its cognate ligand, RANKL, expressed by osteoblasts is essential for osteoclastogenesis [44]. RANKL is a homotrimeric protein displayed on the membrane of osteoblasts, although it may be secreted following cleavage by MMPs 7 or 14, or ADAM (a disintegrin and metalloprotease domain). Suppression of MMP 14-mediated secretion enhances osteoclastogenesis[47,48]. The RANK receptor is a homotrimeric transmembrane protein belonging to the tumour necrosis factor (TNF) receptor superfamily. Following binding of RANKL to RANK, TRAF (TNF receptor-associated factor) adaptor proteins are recruited, with binding sites for TRAF2, TRAF5 and TRAF6 all present on RANK. TRAF6 seems to play a central role in RANK mediated osteoclast formation, and mice deficient in TRAF6 are osteopetrotic whereas TRAF2 and TRAF5 are relatively marginal players in osteoclastogenesis. Signalling via RANK, and these adaptor proteins, activates a number of transcription factors, including NFκB (nuclear factor κB), AP-

1(activator protein 1) and NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin dependent 1) which drive osteoclast differentiation[44]. Osteoprotegrin (OPG) is an endogenous inhibitor of RANKL signalling, functioning as a decoy receptor that binds to RANKL and prevents its association with RANK.

### **5.3 Inflammation in bone infection**

466 Recent Advances in Arthroplasty

Osteoblasts are not terminally differentiated, and some may form osteocytes and become implanted in the bone matrix, eventually ceasing the secretion of osteoid, whilst others undergo apoptosis. Osteocytes are also involved in bone maintenance, detecting stress within the bone through echanosensitive mechanisms located in extensive cellular projections, called canaliculi, that interconnect osteocytes[42]. Osteocytes are thought to respond to mechanical stress by undergoing apoptosis, leading to osteoclast recruitment and differentiation, possibly by alterations in the levels of soluble factors produced by the osteocyte. Candidates include transforming growth factor β(TGF-β), which may suppress

Osteoclasts, which are multinucleate cells derived from the acrophage-monocyte lineage, perform the opposing action of bone matrix removal. These cells express large quantities of a vacuolar-type H+-ATPase on their cell surface, along with chloride channel 7 (ClC 7)enabling localised hydrochloric acid secretion into a closed compartment, known as the resorption lacuna, and subsequent olubilisation of bone mineral. The cell is attached to the bone matrix by a sealing zone membrane to create this compartment, and fusion of acidified vesicles with the plasma membrane contributes further to acid release[45].Following mineral solubilisation, proteolysis of bone matrix proteins is then possible. Cathepsin K is centrally involved in degradation of bone matrix, it is highly expressed by osteoclasts and digests substrates such as collagen and osteonectin. Evidence from knock-out mouse and selective inhibitor experiments indicates that cathepsin L, and MMPs also play a role in degrading bone matrix[46]. Osteoclasts also secrete acid phosphatases, such as tartrate-resistant acid phosphatase(TRAcP), which is used as an osteoclast marker and is activated by cathepsin K cleavage. TRAcP is able to generate reactive oxygen species in addition to having phosphatase activity. The exact cellular function of TRAcP in bone resorption is not well understood, but serum TRAcP levels correlate with bone-resorptive activity, and TRAcPdeficient mice exhibit reduced osteoclastic bone resorption and increased bone

The balance of activity between these two cell types is crucial to maintaining the proper homeostasis of bone turnover, and any shift in the relative levels of osteoblast and osteoclast activity can result in bone pathology. Infection with a pathogen such as S. aureus is capable of stimulating such a shift, mediated in part by induction of an inflammatory response. There is intimate interaction between the two cell types, with osteoblasts interpreting the majority of extracellular signals and subsequently modulating osteoclast differentiation and

Interaction between the RANK (receptor activator for nuclear factor κB) receptor, expressed by osteoclast precursors, and its cognate ligand, RANKL, expressed by osteoblasts is essential for osteoclastogenesis [44]. RANKL is a homotrimeric protein displayed on the membrane of osteoblasts, although it may be secreted following cleavage by MMPs 7 or 14, or ADAM (a disintegrin and metalloprotease domain). Suppression of MMP 14-mediated secretion enhances osteoclastogenesis[47,48]. The RANK receptor is a homotrimeric transmembrane protein belonging to the tumour necrosis factor (TNF) receptor superfamily. Following binding of RANKL to RANK, TRAF (TNF receptor-associated factor) adaptor proteins are recruited, with binding sites for TRAF2, TRAF5 and TRAF6 all present on RANK. TRAF6 seems to play a central role in RANK mediated osteoclast formation, and mice deficient in TRAF6 are osteopetrotic whereas TRAF2 and TRAF5 are relatively marginal players in osteoclastogenesis. Signalling via RANK, and these adaptor proteins, activates a number of transcription factors, including NFκB (nuclear factor κB), AP-

osteoclastogenesis when produced by healthy osteocytes[43,44].

mineralization.

function[44].

A number of host cytokines play a significant role in the pathogenesis of osteomyelitis, and there is strong evidence that production of these cytokines is induced by staphylococcal infection of bone, and that they directly contribute to bone destruction. In particular, the inflammatory cytokines tumour necrosis factor α (TNFα), interleukin 1 (IL-1) and IL-6 seem to be especially important in bone physiology and pathology. In patients with acute osteomyelitis, plasma levels of TNFα, IL-1β (the secreted form of IL-1) and IL-6 are all elevated. High levels of IL-1β, IL-6 and TNFα are also found in the synovial fluid of patients with septic arthritis. Interestingly, specific polymorphisms in the IL-1α and IL-6 genes have recently been found to be associated with an increased risk of osteomyelitis in the Greek population[49]. The most recently identified member of the IL-1 cytokine superfamily is IL-33, has been shown to expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells[50].

A number of animal models of *S. aureus* osteomyelitis reveal that bone infection can lead to elevated levels of these cytokines both locally and systemically. Increased levels of IL-1β have been measured in the tibiae of 22-month-old rats experimentally implanted with *S. aureus* infected needles, and the same animals have increased circulating levels of IL-6[51]. In a murine osteomyelitis model, bone levels of IL-1β and IL-6 are significantly increased in the early post-infection period, with TNFα rising later during the infection. The local source of these cytokines is not fully clear. Production of IL-1β can be induced in human osteoblastlike cell lines by a variety of stimuli, including TNFα. However, infection of primary mouse osteoblasts with *S.aureus* results in increased transcription, but not increased protein synthesis or secretion of IL-1β. TNFα is detectable only at low levels in human osteoblasts derived from mesenchymal stem cells and the osteosarcoma cell line MG63.Infiltrating immune cells may therefore be a more likely source of IL-1β and TNFα in bone in response to infection. IL-6 however, is produced by osteoblasts in response to a variety of signals, including infection with *S. aureus* .

These cytokines have potent effects on the process of bone remodelling, and are strongly implicated in the pathology of osteomyelitis. Cell culture models support the view that IL-1 and TNFα stimulate the proliferation and differentiation of osteoclast progenitors into mature osteoclasts in the presence of osteoblasts. TNFα and IL-1β also stimulate osteoclastmediated bone resorption, a process which may also require the presence of osteoblasts. Similarly, IL-6 increases bone resorption activity and osteoclast number in cultured mouse calvariae, and stimulates osteoclast differentiation in the presence of osteoblasts. In vivo, local administration of IL-1 and TNF antagonists in a non-human primate model of periodontitis results in significant reduction of osteoclast formation and bone destruction. Intravenous administration of TNFα and IL-1 in mice stimulates bone resorption in a dosedependent fashion, and deletion of the murine IL-1R, TNF-R1 and TNF-R2 receptors and of caspase-1 significantly decreases osteoclast number and the area of bone resorption in calvariae following lipopolysaccharide (LPS) injection.

IL-1β and TNFα also inhibit the differentiation of mesenchymal stem cells into osteoblastlike cells, and suppress the accompanying mineralisation and increased expression of

Staphylococcus Infection Associated with Arthroplasty 469

and subsequent phosphorylation and activation of STAT (signal transducers and activators of transcription) family transcription factors. IL-6 mediates bone resorption indirectly, and has no effect on isolated osteoclasts and IL-6 induction of osteoclastogenesis is dependent on

It is clear that these cytokines have a prominent role in modulating bone turnover, and perturbation of their levels can have profound effects on this process. Although some mechanistic details are currently lacking, there is strong evidence that *S. aureus* infection of bone initiates local and systemic production of TNFα, IL-1 and IL-6 via host PRRs. Elevated levels of these cytokines then shift the homeostatic balance of bone turnover, increasing osteoclast differentiation and bone resorption and iminishing osteoblast-mediated bone

In addition to staphylococcal induction of inflammatory mediators that modulate the actions of osteoblasts and osteoclasts, bacteria of this genus are involved in more direct interactions with bone cells. Invasion and persistence of *S. aureus* in 'non-professional phagocytic' host cells in vitro has been described for many different cell types, including epithelial cells, endothelial cells and keratinocytes[59]. In cell culture systems, *S. aureus* is able to invade cultured osteoblasts from murine, human and embryonic chick sources, and *S. epidermidis* is also able to invade and grow within cultured osteoblasts. Electron microscopy has demonstrated the presence of bacteria within osteoblasts and osteocytes of embryonic chicks following injection with *S. aureus,* indicating that internalisation by bone cells also occurs in vivo. Intracellular bacteria inside osteoblasts and osteocytes in a patient with recurrent, long-term osteomyelitis of the fibula have been visualised by light and electron microscopy. More recently Stoodley et al. [60]have demonstrated *S. aureus* biofilms in an infected total joint arthroplasty. Although not reported in this paper the authors also identified *S. aureus* within host cells (personal communications, Stoodley). Thus the suggestion that internalisation of *S. aureus* by bone cells in vivo provides a protective niche for the bacterium, where it is shielded from immune effector mechanisms and antibiotics, may help to explain persistent cases of osteomyelitis. However, the true importance of

the expression of the IL-6 receptor by osteoblasts, but not osteoclast progenitors.

matrix production and mineralisation, thereby driving bone destruction.

intracellular staphylococci in clinical osteomyelitis has yet to be established.

*S. aureus* requires fibronectin-binding proteins (FnBPs) expressed on the surface of the bacterium to enable uptake by osteoblasts, and many other cell types. These proteins belong to a group of adhesions known as MSCRAMMs (microbial surface components recognising adhesive matrix molecules), which bind a range of extracellular matrix proteins including fibronectin, fibrinogen, collagen, elastin and bone sialoprotein. Mutants deficient in the two FnBPs, FnBPA and FnBPB invade host cells very poorly. Invasion is dependent on fibronectin binding by these proteins, and on the host cell integrin α5β1 receptor. *S. aureu*s binds to fibronectin via FnBPs displayed on the bacterial surface, and fibronectin serves as a bridging molecule to the integrin α5β1 which acts as a "phagocytic"receptor. Alternative uptake mechanisms do exist in certain cell types, however, as *S. aureus* is still able to invade primary keratinocytes in the absence of FnBPs and uptake is not inhibited by blockade of integrin α5β1 binding to fibronectin. The mechanism of invasion also differs between *S. aureus* and *S. epidermidis* and the latter does not gain entry via the fibronectin-integrin α5β1

The level of expression of the alternative sigma factor, σB, affects *fnbA* expression and the fibronectin binding ability of *S. aureus* strains and correlates with the level of internalization

**5.4 Staphylococcal invasion of bone cells** 

mechanism[61].

alkaline phosphatase and procollagen I genes, although only TNFα inhibits osteonectin and osteopontin gene expression[52]. TNFα also decreases production of type I collagen and osteocalcin, and of alkaline phosphatase in a variety of osteoblast cell culture and bone tissue explant models, thereby reducing matrix deposition and mineralisation.

Surface-associated material (SAM) from *S. aureus* stimulates bone resorption and osteoclast formation, and blockade of IL-1 or TNFα signalling completely abolishes this bone resorption activity. Neutralisation of TNFα and IL-6 fully abolishes SAM-stimulated osteoclastogenesis, with antagonism of IL-1 having only a partial effect.

The effect of this SAM on osteoclast formation and stimulation of resorption does not require co-culture with osteoblasts, and does not require RANKL signaling[53]. *S.epidermidis*  surface material can also induce bone resorption, by a mechanism that is strongly dependent on TNFα and, to a lesser extent, IL-1.Induction and release of these cytokines in response to pathogen-associated molecules involves two main classes of pattern recognition receptors (PRRs), the Toll-like receptors (TLRs) and NOD-like receptors (NLRs). The production of TNFα and IL-6 by murine macrophages in response to *S. aureus* cell wall preparations is dependent on TLR2, and TLR2-deficient mice exhibit reduced survival of intravenous *S. aureus* infections compared to wild-type counterparts. Signalling through TLRs, in response to microbial ligands such as LPS, 'primes' the cell for IL-1β production by inducing expression of the inactive, pro-form of the cytokine[54,55].IL-1β is synthesised as a 31-kDa precursor molecule, and is processed to produce a 17-kDa active molecule by caspase-1. Caspase-1 activation, and subsequent processing and release of active IL-1β involves assembly of a multiprotein complex known as the inflammasome. This complex consists of caspase-1, the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) and one of several NLR proteins, of which four are known to associate with inflammasomes[56].Each NLR responds to different activating signals, and although the exact recognition steps remain to be elucidated, reported stimuli include flagellin, anthrax lethal toxin, and muramyl dipeptide. A broad range of stimuli for NLRP3 (NLR family pyrin domain containing 3) have been reported, including *S. aureus*. Although NLRP3 and ASC are essential for IL-1β secretion by murine macrophages in response to *S. aureus,* the stimulating signal is as yet unknown, and deletionof the α-, β- and γ-toxins does not perturb production of the cytokine. The inflammasome is involved in cell death in response to bacterial invasion and although invasion of murine osteoblasts by *S. aureus* induces apoptosis, it is not established whether the inflammasome is involved.

Signalling in response to IL-1 and TNF binding of to their respective receptors leads to eventual activation of the NFκB transcription factor and JNK (c-Jun N-terminal kinase) and p38 MAPK(mitogen-activated protein kinase) signaling[57,58]. Studies with knockout mice have shown that at least one of the p50 or p52 NFκB subunits is required for IL-1-induced osteoclast formation and resorptive activity, indicating that much of the osteoclastogenic activity of IL-1 is dependent on NFκB. TNFα binds to two receptors, TNF type I (TNF-R1) and type II (TNF-R2) receptor which differ in their signaling mechanisms although there is substantial signalling crosstalk between the two receptors. Interestingly, the *S. aureus*  virulence factor protein A, in addition to possession of immunoglobulin G-binding activity, is able to bind to the TNF-R1 receptor and stimulates downstream signalling and inflammation.

TNFα mediates the osteoclastogenic activity of RANKL. TNFα production by osteoclast progenitors is induced by RANKL and stimulates osteoclast differentiation in an autocrine manner. Signal transduction involves the activation of Janus family (JAK) tyrosine kinases

alkaline phosphatase and procollagen I genes, although only TNFα inhibits osteonectin and osteopontin gene expression[52]. TNFα also decreases production of type I collagen and osteocalcin, and of alkaline phosphatase in a variety of osteoblast cell culture and bone

Surface-associated material (SAM) from *S. aureus* stimulates bone resorption and osteoclast formation, and blockade of IL-1 or TNFα signalling completely abolishes this bone resorption activity. Neutralisation of TNFα and IL-6 fully abolishes SAM-stimulated

The effect of this SAM on osteoclast formation and stimulation of resorption does not require co-culture with osteoblasts, and does not require RANKL signaling[53]. *S.epidermidis*  surface material can also induce bone resorption, by a mechanism that is strongly dependent on TNFα and, to a lesser extent, IL-1.Induction and release of these cytokines in response to pathogen-associated molecules involves two main classes of pattern recognition receptors (PRRs), the Toll-like receptors (TLRs) and NOD-like receptors (NLRs). The production of TNFα and IL-6 by murine macrophages in response to *S. aureus* cell wall preparations is dependent on TLR2, and TLR2-deficient mice exhibit reduced survival of intravenous *S. aureus* infections compared to wild-type counterparts. Signalling through TLRs, in response to microbial ligands such as LPS, 'primes' the cell for IL-1β production by inducing expression of the inactive, pro-form of the cytokine[54,55].IL-1β is synthesised as a 31-kDa precursor molecule, and is processed to produce a 17-kDa active molecule by caspase-1. Caspase-1 activation, and subsequent processing and release of active IL-1β involves assembly of a multiprotein complex known as the inflammasome. This complex consists of caspase-1, the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) and one of several NLR proteins, of which four are known to associate with inflammasomes[56].Each NLR responds to different activating signals, and although the exact recognition steps remain to be elucidated, reported stimuli include flagellin, anthrax lethal toxin, and muramyl dipeptide. A broad range of stimuli for NLRP3 (NLR family pyrin domain containing 3) have been reported, including *S. aureus*. Although NLRP3 and ASC are essential for IL-1β secretion by murine macrophages in response to *S. aureus,* the stimulating signal is as yet unknown, and deletionof the α-, β- and γ-toxins does not perturb production of the cytokine. The inflammasome is involved in cell death in response to bacterial invasion and although invasion of murine osteoblasts by *S. aureus* induces apoptosis, it is not established whether the inflammasome is involved. Signalling in response to IL-1 and TNF binding of to their respective receptors leads to eventual activation of the NFκB transcription factor and JNK (c-Jun N-terminal kinase) and p38 MAPK(mitogen-activated protein kinase) signaling[57,58]. Studies with knockout mice have shown that at least one of the p50 or p52 NFκB subunits is required for IL-1-induced osteoclast formation and resorptive activity, indicating that much of the osteoclastogenic activity of IL-1 is dependent on NFκB. TNFα binds to two receptors, TNF type I (TNF-R1) and type II (TNF-R2) receptor which differ in their signaling mechanisms although there is substantial signalling crosstalk between the two receptors. Interestingly, the *S. aureus*  virulence factor protein A, in addition to possession of immunoglobulin G-binding activity, is able to bind to the TNF-R1 receptor and stimulates downstream signalling and

TNFα mediates the osteoclastogenic activity of RANKL. TNFα production by osteoclast progenitors is induced by RANKL and stimulates osteoclast differentiation in an autocrine manner. Signal transduction involves the activation of Janus family (JAK) tyrosine kinases

tissue explant models, thereby reducing matrix deposition and mineralisation.

osteoclastogenesis, with antagonism of IL-1 having only a partial effect.

inflammation.

and subsequent phosphorylation and activation of STAT (signal transducers and activators of transcription) family transcription factors. IL-6 mediates bone resorption indirectly, and has no effect on isolated osteoclasts and IL-6 induction of osteoclastogenesis is dependent on the expression of the IL-6 receptor by osteoblasts, but not osteoclast progenitors.

It is clear that these cytokines have a prominent role in modulating bone turnover, and perturbation of their levels can have profound effects on this process. Although some mechanistic details are currently lacking, there is strong evidence that *S. aureus* infection of bone initiates local and systemic production of TNFα, IL-1 and IL-6 via host PRRs. Elevated levels of these cytokines then shift the homeostatic balance of bone turnover, increasing osteoclast differentiation and bone resorption and iminishing osteoblast-mediated bone matrix production and mineralisation, thereby driving bone destruction.

### **5.4 Staphylococcal invasion of bone cells**

In addition to staphylococcal induction of inflammatory mediators that modulate the actions of osteoblasts and osteoclasts, bacteria of this genus are involved in more direct interactions with bone cells. Invasion and persistence of *S. aureus* in 'non-professional phagocytic' host cells in vitro has been described for many different cell types, including epithelial cells, endothelial cells and keratinocytes[59]. In cell culture systems, *S. aureus* is able to invade cultured osteoblasts from murine, human and embryonic chick sources, and *S. epidermidis* is also able to invade and grow within cultured osteoblasts. Electron microscopy has demonstrated the presence of bacteria within osteoblasts and osteocytes of embryonic chicks following injection with *S. aureus,* indicating that internalisation by bone cells also occurs in vivo. Intracellular bacteria inside osteoblasts and osteocytes in a patient with recurrent, long-term osteomyelitis of the fibula have been visualised by light and electron microscopy. More recently Stoodley et al. [60]have demonstrated *S. aureus* biofilms in an infected total joint arthroplasty. Although not reported in this paper the authors also identified *S. aureus* within host cells (personal communications, Stoodley). Thus the suggestion that internalisation of *S. aureus* by bone cells in vivo provides a protective niche for the bacterium, where it is shielded from immune effector mechanisms and antibiotics, may help to explain persistent cases of osteomyelitis. However, the true importance of intracellular staphylococci in clinical osteomyelitis has yet to be established.

*S. aureus* requires fibronectin-binding proteins (FnBPs) expressed on the surface of the bacterium to enable uptake by osteoblasts, and many other cell types. These proteins belong to a group of adhesions known as MSCRAMMs (microbial surface components recognising adhesive matrix molecules), which bind a range of extracellular matrix proteins including fibronectin, fibrinogen, collagen, elastin and bone sialoprotein. Mutants deficient in the two FnBPs, FnBPA and FnBPB invade host cells very poorly. Invasion is dependent on fibronectin binding by these proteins, and on the host cell integrin α5β1 receptor. *S. aureu*s binds to fibronectin via FnBPs displayed on the bacterial surface, and fibronectin serves as a bridging molecule to the integrin α5β1 which acts as a "phagocytic"receptor. Alternative uptake mechanisms do exist in certain cell types, however, as *S. aureus* is still able to invade primary keratinocytes in the absence of FnBPs and uptake is not inhibited by blockade of integrin α5β1 binding to fibronectin. The mechanism of invasion also differs between *S. aureus* and *S. epidermidis* and the latter does not gain entry via the fibronectin-integrin α5β1 mechanism[61].

The level of expression of the alternative sigma factor, σB, affects *fnbA* expression and the fibronectin binding ability of *S. aureus* strains and correlates with the level of internalization

Staphylococcus Infection Associated with Arthroplasty 471

because whilst the murine septic arthritis model is well established and standardised a number of different models have been developed for osteomyelitis and the relevance of specific virulence factors to bone implant infections or osteomyelitis appears to be dependent on the particular model used. For example the collagen adhesin Cna has been shown to contribute to osteomyelitis by some workers but not by others and has been reported not to be important in orthopaedic device infections. The role of FnBPs has not been directly assessed in a model of osteomyelitis, but comparison of *S. aureus* strains with and without fibronectin-binding activity in a mouse osteomyelitis model suggests that fibronectin-binding strains may give rise to more severe bone infections. In the septic arthritis model, *S. aureus fnbA fnbB* mutants show no reduction in severity of arthritis, in contrast with *clfA clfB* mutants lacking the fibrinogen-binding clumping factors. However, the presence of the *fnb* genes results in greater weight loss and mortality, as well as higher serum levels of IL-6, indicating a role for FnBPs in the systemic inflammatory response. One area of research that has received surprisingly little attention is that of the direct action of virulence factors on bone and bone cells. Work in our own laboratory has shown that *S.aureus* and *S. epidermidis* produce surface-associated proteins that can stimulate bone breakdown in an in vitro assay. These surface-associated proteins and capsular material appear to promote the formation and activation of the boneresorbing osteoclast. Interestingly, a proportion of the population have antibodies that can block the action of the *S. aureus* proteins and prevent bonebreakdown. The identity of the protein(s) in these

Variant forms of *S. aureus*, known as small colony variants (SCVs), are associated with infections of bone and joint that may be particularly persistent, recurrent and refractory to antibiotic treatment[65]. These bacteria are mutant forms of *Staphylococcus* that may have an

SCV forms of coagulase-negative staphylococci, including *S. epidermidis*, *S. lugdunensis* and *S. capitis* have also been isolated from a range of infections. The SCV phenotype is characterised by slow growth, with colonies around 10-fold smaller than wild-type forms, often with decreased pigmentation, increased aminoglycoside resistance and some reports of reduced haemolytic activity[66]. The nature of these phenotypes can cause difficulty in detection and identification of the bacteria, and may contribute to an underestimation of the clinical prevalence of SCVs [67]. These phenotypes usually result from auxotrophy for hemin, menadione or thymidine and can be reversed by supplementation with these molecules. Mutations in the *hemB* and *menD* genes produce hemin and menadione auxotrophic strains with typical SCV phenotypes, and give rise to disruption of electron transport which is the basis of the growth deficiency, increased minoglycoside resistance and other phenotypes. SCVs can be selected for with gentamicin in vitro, and there is evidence that antibiotic therapy, in particular use of gentamicin beads, which are used in addition to debridement and systemic antibiotic therapy for osteomyelitis may select for SCVs in clinical situations. In a cohort of fourteen patients with confirmed *S. aureus*  osteomyelitis, SCVs were isolated only from those four that had received gentamicin bead therapy, with the remaining ten patients harbouring normal *S. aureus* strains. Of the four SCVs, three were auxotrophic for hemin, and one for menadione. Only the patients harbouring SCVs had recurrent infections, although only patients whose gentamicin bead

mixtures which cause bone destruction has not been elucidated.

adaptive advantage enabling persistent bone colonisation.

**5.6 Small colony variants** 

of bacteria by osteoblasts suggesting that σB-mediated up-regulation of FnBP expression may facilitate invasion[62].

Integrin α5β1-mediated uptake of *S. aureus* requires remodelling of the actin cytoskeleton. The integrin-linked kinase, ILK, provides a link between α5β1 and the cytoskeleton, and interacts with the cytoplasmic domains of β integrins and is subsequently activated. ILK activity is required for internalisation of *S. aureus* by epithelial cells. Recruitment of focal adhesion proteins, including the adaptor protein paxillin and the focal adhesion kinase, FAK, follows. Upon infection of HEK293T cells with *S. aureus* there is also recruitment of focal adhesion proteins, such as tensin, zyxin and vinculin to the site of bacterial attachment. FAK is recruited and tyrosine phosphorylated, and FAK-deficient cells are able to internalise *S. aureus* much less efficiently. Phosphorylation of downstream substrates of FAK, including cortactin, which is involved in actin cytoskeletal organisation, occurs during invasion, and interference with cortactin also reduces internalisation. So, signalling downstream of the integrin α5β1 receptor, involving ILK and FAK, is important for *S. aureus* invasion, at least in certain cell types.

Physical contact between *S. aureus* and osteoblasts induces host cell expression of tumour necrosis factor apoptosis inducing ligand (TRAIL) . TRAIL is a member of the TNF cytokine family, and binds to two death domain -containing receptors, TRAIL receptors 1 and 2, which once activated recruit the FADD (Fas-associated protein with death domain) adaptor protein which in turn activates caspases 8 and −10 and commits the cell to an apoptotic pathway[63]. TRAIL produced by *S. aureus*-infected osteoblasts induces caspase-8 activation and apoptosis in cultured osteoblasts. Uninfected osteoblasts cultured alongside infected cells also express TRAIL[64]. TRAIL can induce apoptosis in human osteoclasts via TRAIL receptor 2, and also inhibits osteoclast differentiation. It is therefore possible that apoptosis of bone cells infected with *S. aureus*, and potentially of neighbouring uninfected cells may contribute to bone loss in osteomyelitis .

Growing experimental support indicates that staphylococcal invasion of osteoblasts, most likely via the FnBP-fibronectin-integrin α5β1 bridging mechanism in the case of *S. aureus*, may play a role in the pathogenesis of bone infections. This intracellular location may provide a protected environment for bacteria, aiding prolonged persistence by enabling evasion of antimicrobials and host immune mechanisms and possibly contributing to bone damage by inducing apoptosis of infected cells.

### **5.5 Staphylococcal virulence determinants**

A number of animal models of bone implant infection, osteomyelitis and septic arthritis have been developed which have enabled the role of specific virulence factors in infections to be determined. As mentioned at the outset there are a number of routes of bone infection, i.e. haematogenous, contiguous and direct infection of bone, and models have been developed to mimic each of these routes of infection. This is important since the range of environments experienced by the bacterium differs for each route and hence the virulence factors that are involved in pathology may be different for each route of infection. The septic arthritis model developed by Tarkowski and colleagues in conjunction with defined isogenic mutants deficient in one or more virulence determinants, or with neutralising antibodies to virulence factors has proven to be particularly useful in elucidating the role of specific virulence determinants and host factors in bone infections. This model has shown that there is a plethora of virulence determinants involved in *S. aureus* septic arthritis. some of which are also involved in osteomyelitis. However, there is some controversy in this area

of bacteria by osteoblasts suggesting that σB-mediated up-regulation of FnBP expression

Integrin α5β1-mediated uptake of *S. aureus* requires remodelling of the actin cytoskeleton. The integrin-linked kinase, ILK, provides a link between α5β1 and the cytoskeleton, and interacts with the cytoplasmic domains of β integrins and is subsequently activated. ILK activity is required for internalisation of *S. aureus* by epithelial cells. Recruitment of focal adhesion proteins, including the adaptor protein paxillin and the focal adhesion kinase, FAK, follows. Upon infection of HEK293T cells with *S. aureus* there is also recruitment of focal adhesion proteins, such as tensin, zyxin and vinculin to the site of bacterial attachment. FAK is recruited and tyrosine phosphorylated, and FAK-deficient cells are able to internalise *S. aureus* much less efficiently. Phosphorylation of downstream substrates of FAK, including cortactin, which is involved in actin cytoskeletal organisation, occurs during invasion, and interference with cortactin also reduces internalisation. So, signalling downstream of the integrin α5β1 receptor, involving ILK and FAK, is important for *S. aureus* invasion, at least

Physical contact between *S. aureus* and osteoblasts induces host cell expression of tumour necrosis factor apoptosis inducing ligand (TRAIL) . TRAIL is a member of the TNF cytokine family, and binds to two death domain -containing receptors, TRAIL receptors 1 and 2, which once activated recruit the FADD (Fas-associated protein with death domain) adaptor protein which in turn activates caspases 8 and −10 and commits the cell to an apoptotic pathway[63]. TRAIL produced by *S. aureus*-infected osteoblasts induces caspase-8 activation and apoptosis in cultured osteoblasts. Uninfected osteoblasts cultured alongside infected cells also express TRAIL[64]. TRAIL can induce apoptosis in human osteoclasts via TRAIL receptor 2, and also inhibits osteoclast differentiation. It is therefore possible that apoptosis of bone cells infected with *S. aureus*, and potentially of neighbouring uninfected cells may

Growing experimental support indicates that staphylococcal invasion of osteoblasts, most likely via the FnBP-fibronectin-integrin α5β1 bridging mechanism in the case of *S. aureus*, may play a role in the pathogenesis of bone infections. This intracellular location may provide a protected environment for bacteria, aiding prolonged persistence by enabling evasion of antimicrobials and host immune mechanisms and possibly contributing to bone

A number of animal models of bone implant infection, osteomyelitis and septic arthritis have been developed which have enabled the role of specific virulence factors in infections to be determined. As mentioned at the outset there are a number of routes of bone infection, i.e. haematogenous, contiguous and direct infection of bone, and models have been developed to mimic each of these routes of infection. This is important since the range of environments experienced by the bacterium differs for each route and hence the virulence factors that are involved in pathology may be different for each route of infection. The septic arthritis model developed by Tarkowski and colleagues in conjunction with defined isogenic mutants deficient in one or more virulence determinants, or with neutralising antibodies to virulence factors has proven to be particularly useful in elucidating the role of specific virulence determinants and host factors in bone infections. This model has shown that there is a plethora of virulence determinants involved in *S. aureus* septic arthritis. some of which are also involved in osteomyelitis. However, there is some controversy in this area

may facilitate invasion[62].

in certain cell types.

contribute to bone loss in osteomyelitis .

damage by inducing apoptosis of infected cells.

**5.5 Staphylococcal virulence determinants** 

because whilst the murine septic arthritis model is well established and standardised a number of different models have been developed for osteomyelitis and the relevance of specific virulence factors to bone implant infections or osteomyelitis appears to be dependent on the particular model used. For example the collagen adhesin Cna has been shown to contribute to osteomyelitis by some workers but not by others and has been reported not to be important in orthopaedic device infections. The role of FnBPs has not been directly assessed in a model of osteomyelitis, but comparison of *S. aureus* strains with and without fibronectin-binding activity in a mouse osteomyelitis model suggests that fibronectin-binding strains may give rise to more severe bone infections. In the septic arthritis model, *S. aureus fnbA fnbB* mutants show no reduction in severity of arthritis, in contrast with *clfA clfB* mutants lacking the fibrinogen-binding clumping factors. However, the presence of the *fnb* genes results in greater weight loss and mortality, as well as higher serum levels of IL-6, indicating a role for FnBPs in the systemic inflammatory response.

One area of research that has received surprisingly little attention is that of the direct action of virulence factors on bone and bone cells. Work in our own laboratory has shown that *S.aureus* and *S. epidermidis* produce surface-associated proteins that can stimulate bone breakdown in an in vitro assay. These surface-associated proteins and capsular material appear to promote the formation and activation of the boneresorbing osteoclast. Interestingly, a proportion of the population have antibodies that can block the action of the *S. aureus* proteins and prevent bonebreakdown. The identity of the protein(s) in these mixtures which cause bone destruction has not been elucidated.

### **5.6 Small colony variants**

Variant forms of *S. aureus*, known as small colony variants (SCVs), are associated with infections of bone and joint that may be particularly persistent, recurrent and refractory to antibiotic treatment[65]. These bacteria are mutant forms of *Staphylococcus* that may have an adaptive advantage enabling persistent bone colonisation.

SCV forms of coagulase-negative staphylococci, including *S. epidermidis*, *S. lugdunensis* and *S. capitis* have also been isolated from a range of infections. The SCV phenotype is characterised by slow growth, with colonies around 10-fold smaller than wild-type forms, often with decreased pigmentation, increased aminoglycoside resistance and some reports of reduced haemolytic activity[66]. The nature of these phenotypes can cause difficulty in detection and identification of the bacteria, and may contribute to an underestimation of the clinical prevalence of SCVs [67]. These phenotypes usually result from auxotrophy for hemin, menadione or thymidine and can be reversed by supplementation with these molecules. Mutations in the *hemB* and *menD* genes produce hemin and menadione auxotrophic strains with typical SCV phenotypes, and give rise to disruption of electron transport which is the basis of the growth deficiency, increased minoglycoside resistance and other phenotypes. SCVs can be selected for with gentamicin in vitro, and there is evidence that antibiotic therapy, in particular use of gentamicin beads, which are used in addition to debridement and systemic antibiotic therapy for osteomyelitis may select for SCVs in clinical situations. In a cohort of fourteen patients with confirmed *S. aureus*  osteomyelitis, SCVs were isolated only from those four that had received gentamicin bead therapy, with the remaining ten patients harbouring normal *S. aureus* strains. Of the four SCVs, three were auxotrophic for hemin, and one for menadione. Only the patients harbouring SCVs had recurrent infections, although only patients whose gentamicin bead

Staphylococcus Infection Associated with Arthroplasty 473

Establishing the presence of acute infection or, in the presence of a draining sinus, chronic infection, is uncomplicated. In these situations, testing may be limited to that needed to establish the microbiologic diagnosis. Chronic infection manifested as localized joint pain alone poses more diagnostic difficulty, warranting additional testing. The criteria for interpreting laboratory and imaging findings in patients with a prosthetic joint are distinct from those applied in patients with a native joint. In addition to establishing the diagnosis, the identification of the involved organism or organisms and their antimicrobial susceptibility (i.e., on the basis of cultures of synovial fluid, periprosthetic tissue, the implant, or a combination of

**C-Reactive Protein**—In the absence of underlying inflammatory conditions, CRP measurement is the most useful preoperative blood test for detecting infection associated with a prosthetic joint. CRP testing has a sensitivity of 73 to 91% and a specificity of 81 to 86% for the diagnosis of prosthetic-knee infection with the use of a cutoff point of 13.5 mg per liter or more[68,69]. It has a sensitivity of 95% and a specificity of 62% for the diagnosis of prosthetichip infection with the use of a cutoff point of more than 5 mg per liter[70]. Although the CRP level and erythrocyte sedimentation rate are elevated after uncomplicated arthroplasty, the CRP level returns to the preoperative level within 2 months, whereas the erythrocyte edimentation rate may remain elevated for several months. A normal CRP level generally indicates an absence of infection, although false negative results may occur in patients who have been treated with antimicrobial agents or who have infection that is caused by low-virulenceorganisms such as *P. acnes*. Elevations in the peripheral -blood leukocyte count and levels of procalcitonin have low sensitivity for

**Imaging**—Plain radiography has low sensitivity and low specificity for detecting infection associated with a prosthetic joint[71]. Periprosthetic radiolucency, osteolysis, migration, or all of these features may be present on radiographs in patients with either infection or aseptic loosening of the prosthesis. Diagnostic studies with the use of computed tomography (CT) or magnetic resonance imaging (MRI) are hampered by artifacts produced by prostheses, although implants that are not ferromagnetic (i.e., titanium or tantalum) are associated with minimal MRI artifacts, and MRI scans of such implants provide good resolution for detecting soft-tissue abnormalities. Bone scans obtained after the administration of technetium-99m –labeled methylene diphosphonate are sensitive for detecting failed implants but nonspecific for detecting infection, and they may remain

Some studies suggest that combined bone and gallium-67 scans are more specific than bone scans alone. However, labeled-leukocyte imaging (e.g., leukocytes labeled with indium-111) combined with bone marrow imaging with the use of technetium -99m–labeled sulfur colloid is more accurate than bone imaging alone, combined bone and gallium-67 imaging, or labeledleukocyte and bone imaging when compared head to head, and it is considered the imaging test of choice when imaging is required[71]. 18F-fluorodeoxyglucose positronemission tomography (PET) has a sensitivity of 82% and a specificity of 87% for the detection of prosthetic-knee or prosthetic-hip infection, on the basis of pooled data from several studies, but it is not widely available[72]. Newer imaging strategies such as scintigraphy with antigranulocyte monoclonal antibodies and hybrid imaging (e.g.,

such cultures) is important in order to guide antimicrobial therapy.

detecting infection.

abnormal for more than a year after implantation.

combined PET and CT) (see Fig. 3)are under investigation.

therapy had failed were included in the study. SCVs have also been isolated from cases of infection of hip prostheses, and intracellular bacteria within host fibroblasts were identified in one of the five instances.

Clinically isolated SCVs with hemin auxotrophy, and defined *hemB* mutants, show enhanced intracellular persistence in a range of human cell types. The basis of this persistence is not established but may involve a number of possible mechanisms. *S. aureus hemB* mutants exhibit enhanced binding to fibrinogen and fibronectin, and transcribe and display more ClfA and FnBP on their surface, which may increase attachment and uptake by host cells. Transcriptional profiling of clinical and defined mutant SCVs reveals increased transcription of genes regulated by σB, including adhesin genes, and down-regulation of exoprotein and toxin genes. The effect of increased σB activity on MSCRAMM expression has been shown to correlate well with osteoblast invasion, adding weight to the argument that σB-mediated upregulation of adhesins increases host cell invasion, at least in vitro, and that increased invasion by SCVs may be partially dependent on this mechanism. It has been argued that reduced production of toxins, particularly haemolysins, by SCVs also contributes to intracellular persistence by reducing the cytotoxic effect on host cells.

In a murine septic arthritis model, a defined stable *hemB* mutant, exhibiting the SCV phenotype, elicited more frequent and severe arthritis than the parental strain despite a reduced bacterial load in the kidney and joints. It has been argued that SCVs are therefore more virulent on a'per organism' basis and that enhanced protease production by *hemB*  mutants may partially explain this. It may be that in clinical infections relatively small numbers of SCVs with enhanced virulence survive within tissues, possibly intracellularly, for extended periods and cause persistent infections. Clinically isolated SCVs are able to revert to the parent phenotype, although to what extent this may play a role in infections, and whether *S. aureus* may 'switch' between states in different in vivo situations is currently unclear.
