Orthopaedic Infections and Microbiology

Benjamin F. Ricciardi, MD

Edward M. Schwarz, PhD

Dr. Schwarz or an immediate family member is a member of a speakers’ bureau or has made paid presentations on behalf of Asahi KASEI Pharma Corporation; serves as a paid consultant to or is an employee of Amedica, Asahi KASEI Pharma Corporation, DePuy, A Johnson & Johnson Company, MedImmune, Musculoskeletal Transplant Foundation, Regeneron, and Telephus Biomedical; has stock or stock options held in LAGeT Inc. and Telephus Biomedical; has received research or institutional support from DePuy, A Johnson & Johnson Company, Eli Lilly, LAGeT, and Telephus; and has received nonincome support (such as equipment or services), commercially derived honoraria, or other non-research-related funding (such as paid travel) from LAGeT and Telephus. Neither Dr. Ricciardi nor any immediate family member has received anything of value from or has stock or stock options held in a commercial company or institution related directly or indirectly to the subject of this chapter.

ABSTRACT

Infections of the musculoskeletal system are common and challenging to treat. In this chapter, we discuss the microbiology of the major classes of adult musculoskeletal infection. Advancements in our knowledge of the interaction of bacteria and the host immune system will be discussed including bacterial evasion of host immunity, formation of biofilm, bacterial resistance, and persistence within host cells and tissues. Finally, we will discuss therapeutic targets for treatment of musculoskeletal infection including novel antimicrobial agents, immunotherapy, local delivery systems, and dispersal agents.

Keywords: Biofilm; Musculoskeletal infection; Prosthetic joint infection; Staphylococcus aureus

Introduction

Infections of the musculoskeletal system can involve bone, soft tissue, and implant-related material. The societal burden of musculoskeletal infection has been increasing over time because of higher rates of medical comorbidities in patients undergoing orthopaedic surgery and increasing bacterial antibiotic resistance. Novel insights into the mechanisms of musculoskeletal infection and immune system evasion will hopefully expand therapeutic opportunities to treat these challenging conditions.

Microbiology of Musculoskeletal Infection

Epidemiology

The overall incidence of infection at 1 year following orthopaedic surgery varies from 0.5% to 1% after total hip or knee replacement, 2% to 4% for instrumented lumbar fusion, and 5% to 10% after open reduction and internal fixation of open tibia fractures. The microbiological profile of musculoskeletal infection varies based on the anatomic site of infection, presence of an implant, involved tissues, and age of the patient (Table 1). The most common types of musculoskeletal infections are briefly discussed below.

Adult Septic Arthritis

There are over 10,000 adult in-patient admissions in the United States per year with a diagnosis of septic arthritis. Patient risk factors include older age, immunocompromised state (diabetes mellitus, chronic liver disease, chronic kidney disease, immunodeficiency, inflammatory arthritis), intravenous drug use, sexually transmitted disease, infection at other anatomic sites, and geographical exposures (Lyme disease). The knee remains the most commonly affected joint, however, followed by the hip, shoulder, and elbow in decreasing order (Table 1).

Adult Osteomyelitis

The most common cause of osteomyelitis outside of the foot and axial skeleton in adults is previous trauma with a high incidence of retained hardware. Vertebral
osteomyelitis is common in patients who are immunocompromised, those with indwelling catheters, previous spinal instrumentation or spine trauma, and systemic bacteremia from another site. Similar to septic arthritis, Staphylococcus aureus is the most common organism in both extremity and vertebral osteomyelitis and represents up to half of all cases with a high incidence of methicillin resistance in certain populations (Table 1).

Table 1 Summary of the Epidemiology of Major Categories of Deep Musculoskeletal Infection

Common Classes of Deep Musculoskeletal Infection	Most Common Anatomic Sites	Most Common Isolated Organisms
Adult septic arthritis	Knee Hip Shoulder Elbow	Staphylococcus aureus (up to 50% of cases) Streptococcus sp. (group B strep most common) Enterococcus sp. Gram-negative sp. (Neisseria gonorrhoeae in young sexually active populations) Mycobacteria (immunocompromised, developing countries) Propionibacterium acnes (shoulder)
Adult osteomyelitis	Foot Spine/pelvis Sites of previous trauma with retained hardware	Staphylococcus sp. (up to 50% of cases) Streptococcus sp. Enterococcus sp. Gram-negative sp. (Escherichia coli, Pseudomonas aeruginosa)
Prosthetic joint infection	Knee Hip Shoulder	Staphylococcus aureus (up to 50% of cases) Staphylococcus epidermidis Other Staphylococcus sp. (Coagulase negative staphylococci) Streptococcus sp. Enterococcus sp. Gram-negative sp. P acnes (most commonly isolated organism in prosthetic shoulder infection)
Diabetic foot infection	—	Polymicrobial infection more common than monomicrobial isolates Gram-negative sp. (P aeruginosa most common) Gram-positive sp. (Staphylococcus aureus most common; streptococcal sp. [Group B Strep]) Anaerobic sp.

Prosthetic Joint Infection

Prosthetic joint replacement is one of the most common procedures performed in the United States. Overall, the incidence of infection ranges from 0.5% to 2% depending on the anatomic site and study examined. The presence of an implant creates some changes to the microbiological profile from native joint infections. Similar to adult septic arthritis and osteomyelitis, S aureus is the most common cause of prosthetic joint infection (PJI) with resistant strains representing up to 50% of these cases depending on the underlying patient population. Unlike adult septic arthritis and osteomyelitis, Staphylococcus epidermidis also has a high prevalence in PJI with methicillin-resistant strains becoming more common in recent years. These two organisms represent over half of all cases of PJI (Table 1).

Diabetic Foot Infection

The incidence of diabetic foot ulcers has been increasing as the population of patients living with diabetes mellitus grows. Microvascular insufficiency, loss of protective sensation, and deformity from altered bony anatomy of the foot can lead to skin ulceration in these patients. Ulcerations create an opportunity for an infection to develop in the underlying soft tissue and bone. Infected diabetic ulcers increase the risk of lower extremity amputation and limb loss. Diabetic foot infections (DFIs) are more likely to be polymicrobial than the other types of infections discussed, and these are more common than monomicrobial isolates. Additionally, there is a higher incidence of gram-negative organisms
isolated from DFI with Pseudomonas aeruginosa typically being the most common (Table 1).

Host Defense Mechanisms Against Infections

Innate Immune Response

Musculoskeletal infections begin with a breach in local host defenses most commonly involving the skin. This leads to both localized infection at the site of injury and systemic dissemination into other tissues via the circulatory or lymphatic systems. Evasion of host immunity by planktonic, or free floating, bacterial cells is the first step to establishing infection. Integral components of host immunity to acute bacterial infection include responses from cells of the innate immune system (neutrophils, macrophages) and complement. Neutrophils use several antimicrobial strategies to kill planktonic bacteria including phagocytosis, oxidative burst, release of proteases, and production of pro-inflammatory cytokines and chemokines.¹ Additionally, neutrophils produce neutrophil extracellular traps (NETs), which consist of nuclear contents expelled with several antimicrobial proteins deposited on chromatin that are released into the extracellular environment.² NETs help inactive virulence factors, immobilize bacteria, provide a favorable environment for complement activation, and promote phagocyte bacterial clearance.³ Deficiencies in neutrophil number or function (eg, chronic granulomatous disease) result in a significant predisposition to invasive bacterial infection frequently with S aureus, further illustrating their primary importance to host defenses.

Macrophages, in particular classically activated (M1) macrophages, are important for bacterial clearance via phagocytosis in the setting of acute infection by direct recognition of bacterial surface targets through macrophage surface receptors or indirectly by recognizing immunoglobulin or complement on the bacterial surface. Macrophages also act as antigen presenting cells and can activate other components of the innate and adaptive immune system through the release of cytokines and chemokines.

Adaptive Immune Response

The importance of the adaptive immune system is less clear than the innate immune system particularly in the setting of staphylococcal infection. For example, results of a 2018 study show that almost half of patients with a previous S aureus skin infection experience a recurrence despite high titers of specific antibodies and memory T-cells.⁴ Subsets of CD4+ T cells important in adaptive immunity include Th1, Th2, and Th17 cells. The role of Th1 cells (defined by production of interferon-γ (IFNγ)] in mediating protection against S aureus infection varies amongst studies including protective, detrimental, or noncontributory roles depending on the specific experimental conditions.⁵ Th17 cells are defined by expression of Rorγt and cytokines IL-17A, IL-17F, and IL-22.⁵ In the setting of infection, these cells are important in epithelial function and mucosal surface immunity to bacterial or fungal infection, and patients with hyper-IgE syndrome with an IL-17 deficiency have increased susceptibility to S aureus infection.⁶ Undermining the appropriate development of Th17 cells may be another mechanism of S aureus pathogenesis and ability to reinfect hosts after previous infection.⁷ Other T-cell subpopulations such as clonotypic TNF/IFNγ-producing γδ T cells may also play a role in cutaneous immunity against S aureus infection in preclinical models.⁴

B-cell-mediated humoral immunity has been successfully used in vaccine-mediated strategies to prevent bacterial infections such as Haemophilus influenzae. S aureus is unique because it is a commensal organism in humans with up to 30% of the population colonized, but also represents the major pathogenic species in many types of infections.⁸ The immunodominant antigens in S aureus infection in humans and mice are the iron-sensing determinant (Isd) proteins IsdA and IsdB, and components of S aureus major autolysin (aminidase and glucosaminidase).⁹ IgG titers against S aureus-associated proteins are common in healthy humans, and make them challenging for diagnostic purposes because of the high rates of colonization and previous S aureus infection.⁹ Additionally, the presence of anti-S aureus antibodies appears to confer only limited protection against future infection, and vaccine strategies have not been effective in preventing deep S aureus infection (see Treatment section).

Evasion of Host Immunity

Bacterial species use different mechanisms to evade host immunity; however, some common patterns are seen. Using S aureus as a model organism given its propensity for musculoskeletal infection, we will illustrate the various ways bacteria evade host immunity. S aureus utilizes a number of cell-wall-anchored and secreted virulence factors and extracellular matrix binding proteins that promote immunoevasion and biofilm formation (Table 2). Biofilm is an aggregate of microorganisms contained within an extracellular matrix. Key steps in this process include counteracting host immune responses such as neutrophil-mediated killing and complement activation, binding to extracellular matrix components including collagen and fibrin, and scavenging nutrients such as iron in the form of heme via the Isd family of proteins.¹⁰

Table 2. Staphylococcus aureus virulence factors

Table 2 Summary of Selected Cell Wall-Anchored and Secreted Proteins Involved in Staphylococcus aureus Pathogenesis

Class of Protein	Examples	Function
MSCRAMM family proteins—cell wall anchored	Clumping factor A and B (ClfA and ClfB) Fibronectin binding protein A and B (FnBpA, FnBpB) Collagen adhesion (CNA)	Binds host extracellular matrix components particularly fibrinogen. Degradation of complement (C3b) Binds extracellular matrix (fibrinogen, fibrin, elastin) Binds extracellular matrix (collagen, complement protein C1q), inhibits complement activation
NEAT motif family—cell wall anchored	IsdA, IsdB, IsdH	Iron/heme uptake and transport, binds integrins/extracellular matrix components, provides resistance to neutrophil killing
Three helical bundle—cell wall anchored	Protein A Staphylococcal complement inhibitor (SCIN)	Binds Fc region of IgG to inhibit opsonophagocytosis; activates platelet aggregation via von Willebrand factor binding; B cell superantigenic activity by cross-linking Fab region of V_H3 bearing IgM; activates TNFR1 Inhibits complement activation by binding C3 convertases on bacterial surface
Hemolysins	α-Hemolysin (α-toxin, H1a), β-hemolysin, γ-hemolysin	Pore-forming toxin (α-, γ-hemolysin), sphingomyelinase (β-hemolysin). Lyse red blood cells, other leukocytes, epithelial/endothelial cells; alters immune cell signaling pathways involved in cell proliferation, immune response, cytokine expression
Leukocidins	LukAB, LukDE, PVL	Pore-forming toxins. Lyse neutrophils, monocytes, macrophages
Enzymes	Autolysin (AtlA) Aureolysin Staphylokinase Nuclease	Aminidase, glucosaminidase subunits. Peptidoglycan hydrolase. Cell separation, generates extracellular DNA in biofilm matrix Protease. Inactivates PSMs, can activate other proteases Activates plasminogen to plasmin. Cleaves complement factor C3b Inactivate neutrophil extracellular traps (NETs)
Phenol-soluble modulins	δ-Hemolysin, PSMα1-α4	Small amphipathic peptides. Lyse neutrophils, break down biofilm matrix, critical for biofilm disassembly
Superantigenic exotoxins	Toxic shock syndrome toxin Staphylococcal enterotoxins (A-E, G)	Stimulate T cells nonspecifically without typical antigenic recognition. Can cause toxic shock syndrome. Activates bone resorption, inhibits host immunity in osteomyelitis. Activate cytokine release, involved in gastroenteritis, sepsis, kidney injury
Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS)	—	Inhibits neutrophil migration and activation; prevents complement activation
MSCRAMM = microbial surface component recognizing adhesive matrix molecules, NEAT = NEAr-iron transporter (NEAT) domains, PSM = phenol-soluble modulin

Staphylococcal Abscess Communities

The formation of soft-tissue abscesses is one hallmark of musculoskeletal infection and promotes bacterial pathogenesis. For example, S aureus is able to form Staphylococcal abscess communities (SACs), which create a favorable environment for the organism to thrive while evading host immune responses. SAC formation is a pathogen-driven process characterized by the production of factors that recruit host immune cells to the vicinity of S aureus infection. A central, replicating group of bacteria develop and are surrounded by a protective pseudocapsule of fibrin that is created by S
aureus coagulase cleavage of fibrinogen.¹¹ A peripheral ring of necrotic neutrophils then forms around the nidus, which are unable to effectively remove the S aureus and contribute to soft-tissue injury.¹¹ Staphylococcal virulence proteins such as nuclease and adenosine synthase A convert NETs into products that induce macrophage cytotoxicity, which may be a significant mechanism in protecting bacteria within the SAC.¹² Eventually, these SACs can rupture, releasing the bacterium into the surrounding tissue.¹⁰

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