Recent Advances in Orthopedics 3 Matthew S Austin, Gregg R Klein, Irving M Shapiro
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Biofilm-related Infections: Orthopedic Considerations and ImplicationsChapter 1

Noreen J Hickok,
Irving M Shapiro,
Javad Parvizi,
Marc Harwood,
Antonia F Chen
 
INTRODUCTION
Orthopedic infection plagues all sites of bone repair and presents serious complications. While infection can occur at any site, this review is limited to infections occurring as a result of joint and spinal surgeries. Within these categories, in general the highest rates will be associated with spine, followed by knee then hip.1–3
Why do infections occur? Clean room techniques, space suits, negative air pressure and stringent sterile procedure are all used to minimize contamination, but some contamination is almost inevitable in the presence of open and exposed tissue.4–7 Unfortunately, in the presence of an implant, many fewer bacteria can establish infection than are normally required.8–10 Contamination during prolonged surgical times, hematogenous spread from other infected sites in the body, environmental contamination during severe trauma or previous infections that have occurred in the bone environment and which re-establish in the presence of an implant,4,5 all contribute to infection during the perisurgical period; this complication remains low but significant. We continue to make small strides in combatting establishment of infection, but the incidence of bacterial antibiotic insensitivity and resistance continues to grow, therefore multifactorial approaches will be required.
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Noreen J Hickok PhD, Professor, Department of Orthopedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
Irving M Shapiro BDS PhD, Professor, Department of Orthopedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
Javad Parvizi MD, Professor, Department of Orthopedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University and Rothman Institute, Philadelphia, Pennsylvania, USA
Marc Harwood MD, Associate Professor, Department of Orthopedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University and Rothman Institute, Philadelphia, Pennsylvania, USA
Antonia F Chen MD MBA, Director of Arthroplasty Research, Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
 
ETIOLOGY OF IMPLANT INFECTIONS
Implant infections, while occurring infrequently, pose special problems because of the avascular nature of the cartilage and bone, the relative hypoxia of the joint and the intervertebral disc, the general poor penetrance of antibiotic into bone, and the attenuated efficacy of antibiotics against implant-associated bacteria.6–11 Prolonged antibiotic prophylaxis has been tested after joint surgery, with no clear advantage conferred by continued treatment after the first postoperative 24 hours. Because of the reduced efficacy of antibiotics against adherent and biofilm bacteria,12,13 spine and more recently joint surgeons have initiated use of supratherapeutic levels of vancomycin (VAN) (1–2 g placed during closure) perioperatively. Vancomycin is only active against Gram positive pathogens such as methicillin sensitive and methicillin resistant Staphylococcus aureus (MSSA and MRSA). Thus, VAN placed in the spinal surgical site during closure, while decreasing infections due to Gram positive bacteria,14,15 may facilitate establishment of infections with Gram negative bacteria.16
At a molecular level, infection in the vicinity of the implant initiates with a bacterial inoculum; these bacteria can be generally cleared by the immune system or administration of appropriate antibiotics. In the presence of an implant, bacteria rapidly adhere to its surface and undergo changes in metabolism and gene expression to result in reduced antibiotic susceptibility.17 Importantly, the implant surface serves as the perfect host for these bacteria as it is rapidly coated with serum proteins upon insertion into the operative site. It is worth noting that the proteins that coat the implant, e.g., fibronectin, collagen, fibrin and other serum proteins, are proteins that are required for effective osseointegration of the implant and are exactly the proteins that facilitate bacterial adherence to the inert implant.18 With such favorable conditions and the ability to establish infection with low bacterial inoculum, the miracle is that infection is so rare. It is our opinion that the ability of the immune system to eradicate the ever-present, albeit usually low level bacterial contaminants plays a key role in determining who will succumb to implant-associated infection and who will be able to have a successful implantation.
Important steps in this implant-associated biofilm formation have been mapped in vitro, generally using media optimized for bacterial growth, such as Trypticase Soy Broth (TSB). Implant-adherent bacteria control colony formation through quorum sensing and biofilm formation. This process leads to a three dimensional network of bacteria that are encased in a polysaccharide slime. This slime contains large amounts of nucleic acids deposited due to bacterial death during biofilm maturation. The biofilms are semipermeable and have been shown to have channels for nutrient streaming. In fact, the maturing biofilm has been compared to a three-dimensional tissue where position may impact survival. Within the biofilm, bacteria show antibiotic recalcitrance because of:
  • Reduced metabolic rate—many antibiotics rely on active protein synthesis or new cell membrane crosslinking for activity; in the presence of reduced proliferation, these processes are minimal
  • Secretion of factors that sequester antibiotics—by this means, bacteria in the biofilm see reduced concentrations of active antibiotics, allowing them to survive
  • Decreased antibiotic diffusion rates in the polysaccharide and protein-rich biofilm—again, the net result is lowered antibiotic concentrations leading to increased survival of biofilm bacteria.19
For all of these reasons, once a biofilm has formed, even in the presence of high levels of antibiotics, biofilm-embedded bacteria are difficult to kill.3
The effect of bacterial adhesion on antibiotic sensitivity is demonstrated by some early experiments of ours: A titanium rod is rapidly colonized upon incubation with S. aureus. When 4 µg/mL VAN is added simultaneously with S. aureus (strain MIC ≤2 µg/mL VAN), planktonic (nonadherent) S. aureus are completely killed. Importantly, a small portion of S. aureus manage to adhere and these adherent S. aureus easily survive the initial 4 µg/mL VAN. Importantly, they can persist even after 1 hour treatment with 100 µg/mL of VAN.20 This finding underlines the inherent difficulty in treating implant-associated infections, i.e., bacterial adherence to an inert surface causes rapid resistance to antibacterial agents.
The next step is the maturation of the biofilm with reintroduction of planktonic (nonadherent) bacteria. Specifically, when the biofilm reaches a critical size, fragments containing bacteria are sloughed off into the surrounding space, seeding new colonies from this original nidus of infection. Planktonic bacteria, whether from the original infection, or biofilm, can adhere to the bone extracellular matrix (ECM) through specialized binding proteins such as the S. aureus fibronectin-binding protein A21,22 that binds to fibronectin in its type 1 repeats, or the fibrin binding proteins such as clumping factors A or B (clfA, clfB). Bacteria bound to the ECM, most likely infiltrate the bone matrix providing another protected niche until cell death and/or osteolysis again make the infection patent by release of bacteria.
 
IMPLANT INFECTIONS: THE ROLE OF ENVIRONMENT
While this in vitro model reliably predicts many stages of biofilm formation and maturation, it is important to recognize that bacteria present in the physiological environment and bathed in site-specific fluids may behave differently. While both the joints and the spine are prone to infection, the surgical sites around the implants are different enough to warrant separation in our description of factors that influence bacterial behavior.
 
Joint Infection
Periprosthetic joint infection (PJI) is the most serious complication associated with placement of artificial joints and it causes significant bone loss, disability and even death.23 These joints are enclosed in a synovial capsule, which is retained as much as possible after a primary surgery and which allows only limited immune surveillance. Within this capsule, the joints are bathed in the proteoglycan-rich synovial fluid which exists in a relatively low oxygen atmosphere and which is regenerated relatively quickly after surgery. Thus, this environment includes the regenerated synovial fluid, the implant comprised of a polymer/ceramic and metal, and the remnants of the joint including the bone, cartilage and synovium. All of these sites can be colonized by bacteria.
To prevent establishment of infection, many different surfaces have been developed, including ones with controlled release of antibiotics,24,25 different textures,26–29 permanent modification of surfaces with antibiotics or antimicrobial peptides,12,30,31 and materials that innately show some resistance to bacterial colonization. Clinically, controlled release systems, texturing and materials are used; however, only the controlled release systems consistently show marked effects on bacterial colonization of implanted materials, in vitro. The importance of the implant in the establishment of PJI can be teased out using modified permanent antimicrobial implants. In the authors’ work, both in a rat femoral canal periprosthetic infection32 and in a sheep infected osteotomy model,33 a titanium alloy or titanium implant was readily colonized by S. aureus with subsequent display of 4clinical signs of infection. However, if the implant was first modified so that it permanently displayed (no elution) VAN on the implant surface, establishment of infection was prevented. These results emphasized the importance of the immune system as no systemic antibiotics were administered. Thus, the ability of the implant to be colonized may be the most important aspect for the establishment of PJI. The other tissues and fluids in the joint environment take on increasing importance once a bacterial foothold on the implant has been established.
When PJI is finally diagnosed, except in cases where the incidence occurs within the first day or two after surgery, all of the possible natural and implanted surfaces are assumed to be infected. The approach parallels some cancer treatment plans where the affected tissue is excised with large margins and aggressive chemotherapy is initiated to eradicate mobile cells. Due to the difficulty in successfully eradicating bacteria, aggressive antimicrobial treatments such as incubation with Dakin's solution and/or hydrogen peroxide are included during washing. Re-implantation occurs either immediately (one-stage) or an antibiotic controlled-release spacer is used for 6 weeks, followed by prosthesis re-implantation (two-stage).34
Unfortunately, the complexity of the joint plays a role in the persistence of infection. When bacteria proliferate in synovial fluid, they create dense, floating or implant-bound proteinaceous (fibrin-based) biofilms many of which are too large for phagocytosis, markedly decrease expression of virulence factors,35 and become less responsive to antibiotics.36 Specifically, prophylactic antibiotic doses that decrease S. aureus numbers by 8–9 logs under ideal conditions are much less effective when the bacteria are present in floating biofilms within the viscous synovial fluid.36 This attenuated response due to both floating and adherent biofilms, allows persistence of infection and can foster antibiotic resistance. In addition, the presence of antibiotics such as VAN, can foster production of “persisters.” Persisters are bacteria that are similar in genotype to antibiotic-sensitive pathogens, but because of changes in metabolism and expression of surface proteins, enter a persistent state that can allow long-term survival in the presence of antibiotics. As many as 10% of VAN-sensitive S. aureus can enter this persistent state when exposed to high concentrations of the antibiotic.37 The problem becomes manifold once VAN is dissipated and the persisters return to their virulent, proliferative state. Based on recent work, it is possible that once persistence is initiated, the “persisters” are more likely to re-enter the persistent state, ensuring that antimicrobial chemotherapies are more likely to fail.38
The synovial fluid also impacts expression of bacterial virulence factors which determine host-bacterial interactions that will be used to both combat and allow the infection. These factors include toxins that lyse host cells, such as the Panton-Valentine leukocidin,39 the matrix binding proteins mentioned above, toxins and proteins such as the phenol specific modulins which are important for both the aggregation we mentioned earlier as well as activation (and lysis) of neutrophils.40 Importantly, bacteria with mutated virulence factors that ablate binding to ECM and serum proteins show little aggregation and are not pathogenic.41
Virulence factors are largely under control of agr, a master regulator that controls expression of many virulence factors as well as expression of polysaccharide intracellular adhesion, the quorum sensing factor. Formation of the large protein and bacterial aggregates that are characteristic of joint infections results in suppression of agr and of virulence factor expression. Virulence factors cause immune activation on one hand which is desirable, but on the other hand, cause the undesirable immune-mediated 5joint destruction and ultimately sepsis.42 Based on what we measure in synovial fluid, we propose that there is a lag period during which infection is establishing where virulence of the pathogen is attenuated. When bacteria reach a crucial concentration (i.e., the equivalent of biofilm maturation), we would suggest that full virulence occurs accompanied by purulence, osteolysis and immune cell activation and/or destruction.
 
Spinal Infection
Spinal infections are more frequent than joint infections, despite the surgical site being more vascularized than the tissue in the joint space. These elevated rates are associated with long surgical times, hardware presence and the creation of a dead space (≥5 cm3) that is filled with wound exudate so that even the more accessible space is prone to colonization.43 The current standard of care includes irrigation, aggressive debridement of the infected soft tissues and prolonged antibiotic treatment. As spinal stability is critical, infections often must be treated in the presence of the hardware, a particularly difficult task as bacteria adherent to the implant become recalcitrant to antibiotic therapy. These infections often result in extended pain, delayed wound and bone healing, disability and in the worst cases, death.
Unlike the joint, spinal hardware is placed at the interface of the bone and muscle and is thus accessible to blood supply; this placement implies a more normal relative oxygen level; this is important because bacteria such as MSSA and MRSA are facultative anaerobes, that is bacteria that can switch their metabolism from aerobic to anaerobic depending on the environment. MSSA shows greater sensitivity to antibiotics in a normoxic than hypoxic environment (our preliminary data). Thus, the spinal hardware in the more vascularized muscle allows both greater antibiotic sensitivity and increased immune surveillance. Of some concern is the presence of wound exudate which should be similar to serum. Staphylococcus aureus in serum also forms floating biofilms, but the overall size is much smaller and these aggregates show greater antibiotic sensitivity.44,45 It is worth noting that in blood, bacterial sequestration in clots limits infection and enhances elimination; conversely, expression of bacterial proteins that lyse clots cause greater virulence and mortality.46
 
Common Problems
If eradication is not complete, such that a biofilm colony survives again, the most likely site is the implant—conditions are created that foster a chronic infection. Chronic infections may be more frequent than supposed as recent methods of assessing infection attribute many cases of aseptic loosening to infection.47,48 The loosening of the implant would then result from cytokine activation of immune/inflammatory pathways due to bacterial interaction/internalization with resident cells. Whereas in the joint, arthrodesis or amputation can limit the danger of sepsis, in the spine, such infections would be life-threatening.
Specifically, the initiation of chronic infection happens when bacteria associate with the bone matrix or undergo internalization into cells. Within the bone, bacteria associate with porous structures where ingress within the bone is initiated.49 Even without cellular internalization, this population can survive in an indolent state until reactivation causes secretion of virulence factors and bone lysis. An additional site for long-term bacterial ingress is within the resident cells of the bone; this has been described in various tissues 6and has been documented as a source of continuing infection.50,51 It should be noted that these infections will activate cellular Toll-like receptor (TLR) mediated pathways whenever bacteria are present in the joint. The different TLR pathways converge on NF-κB with subsequent production of proinflammatory, immune activating cytokines. On the plus side, these same pathways signal production of the antimicrobial peptide α-defensin which is being used within the synovial fluid as a sensitive indicator of orthopedic infection.52,53
 
PERSPECTIVE
In this article, the authors have detailed the various forms that bacteria adopt to circumvent the current treatments and treatments that some bacteria have the ability to evade. By far, new approaches center on complex controlled release systems or prolonged exposure using a library of antibiotics or antimicrobials, antimicrobial peptides or other antiseptics. The authors suggest that to effectively combat infections, more attention may need to be paid to integration with the immune system. While the first defense will always be strict sterile technique and effective prophylaxis, if that fails, we suggest that integrated, multipronged attacks will be required to reduce the problems of many bacterial phenotypes to ones that can be effectively targeted by available chemotherapeutic agents.
 
CONCLUSION
Infection associated with implantation of an artificial joint or spinal hardware can occur in any patient, and this problem is particularly prevalent and challenging in immunocompromised individuals. When developed, infection can lead to a prolonged period of disability with immense psychosocial and economic cost to the patient and society. The current methods, while immensely successful at preventing infection, need more development for treatment of established infection.
 
ACKNOWLEDGMENT
Research reported in this publication was supported by the National Institutes of Health (NIH) under award numbers R01 AR069119, F32 AR072491, R01 HD06153, and R01 DE019901. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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