The human commensal Staphylococcus epidermidis is the leading cause of orthopaedic prosthetic device infections, resulting in substantial morbidity and cost.11 The ability ofS. epidermidis to adhere to the surface of a prosthetic biomaterial is a pivotal event in the pathogenesis of bio-material-related infection.2 Adherence of S. epidermidis to biomaterials is a multifactorial process.
Initial adherence involves nonspecific and reversible interactions influenced by such factors as bacterial surface charge and hydrophobicity, as well as specific interactions mediated by various adhesins such as AtlE4 and other serum component binding proteins (eg, fibronectin binding protein and fibrinogen binding protein).15 The later stages of biofilm formation are characterized by multicellular aggregation, which is facilitated through the production of polysaccharide intercellular adhesin (PIA),7 a polysaccharide synthesized through enzymes produced by the four-gene operon, icaADBC.1,5 Prior studies have demonstrated the importance of PIA in multicellular aggregation and its role in the pathogenesis of vascular catheter-associated infection.12-14 However, the role of PIA in early adherence is less well characterized and its influence on adherence to orthopaedic biomaterials is unexplored.
We hypothesized PIA facilitates initial adherence to biomaterials, including those used in orthopaedic prosthetic devices.
MATERIALS AND METHODS
We used an in vitro adherence assay to ascertain the adherence of genetically-characterized strains of S. epidermidis to seven biomaterials including those commonly used in orthopaedic prosthetic devices. Strains 1457, 1457 M10, and TM300 were used. 1457 is a clinically relevant, wild-type S. epidermidis isolate that produces a PIA-dependent biofilm and has been shown to be virulent in several animal models.9,10,13,14 Strain 1457 M10 is an isogenic icaA Tn917 mutant lacking the ability to produce a functional biofilm.8 In vivo models of intravascular catheter infection have shown it is less virulent than its isogenic parental strain 1457.13,14 TM300, a PIA-negative strain of S. carnosus, was used as a negative control.3
We used seven biomaterials in the adherence assay: zirconia, ultra-high molecular weight polyethylene (UHMWPE), polymethylmethacrylate (PMMA), cobalt chromium, titanium, silas-tic intravenous catheters, and stainless steel. The surface area of the biomaterial samples was determined in order to standardize adherence measurements and compare one biomaterial to another. Cylindrical, 1 cm long sections of each biomaterial were utilized. The metallic biomaterials (titanium, stainless steel, and cobalt chromium) were sterilized by a steam autoclave. In order to preserve the physical properties of the other biomaterials (PMMA, UHMWPE, zirconia, silastic) sterilization was achieved through ethanol washing.
S. epidermidis strains 1457, 1457 M10, and TM300 were grown overnight (with shaking) in tryptic soy broth (TSB) (Difco, Sparks, MD) to a concentration of 1 × 107 colony forming units (CFU). Two ml aliquots of the broth culture were transferred into 5 mL polystyrene test tubes (Becton Dickinson, Franklin Lakes, NJ) containing the biomaterial sample, one bio-material per tube. The samples were then rocked in the bacterial suspensions on a Labquake shaker (LabIndustries Inc, Berkley, CA) at room temperature for 1 hour. After this incubation period, the biomaterials were removed from the suspensions and placed in 1.5 mL microcentrifuge tubes containing 1 mL of phosphate buffered saline (PBS). These tubes were vortexed at full speed for 1 minute to release the attached cells from the biomaterials. Serial dilutions were then performed and the cells were plated onto Brain Heart Infusion (BHI) (Difco) agar. All tests were performed in triplicate and repeated a minimum of three times.
Statistical analysis was performed using the nonparametric Kruskal Wallis test (GraphPad Prism 4.02, San Diego, CA). Comparisons yielding a p-value of ≤ 0.05 were regarded as significant. Since the surface area of each biomaterial was different, the data were expressed as CFU/mm2 when performing biomaterial-to-biomaterial comparisons.
Compared to the isogenic, PIA-negative mutant 1457 M10, the PIA-positive wild type strain 1457 exhibited greater adherence (p < 0.05) to all biomaterials tested except titanium and PMMA (Fig 1). Additionally, S. epidermidis 1457 exhibited substantially greater adherence (p <0.01) to all tested biomaterials compared to the negative control, S. carnosus TM300. There was no notable difference in the level of adherence to the non-metallic biomaterials (UHMWPE, PMMA, zirconia, and silastic) between PIA-negative S. epidermidis 1457 M10 and S. carnosus TM300. However, S. epidermidis 1457 M10 exhibited greater adherence (p < 0.05) to the metallic bio-materials (titanium, stainless steel, cobalt chromium) than the negative control strain, S. carnosus TM300.
For the PIA-positive S. epidermidis 1457, adherence was greatest to stainless steel and least to PMMA (Fig 2). PIA-negative S. epidermidis 1457 M10 exhibited its greatest adherence to titanium and its least adherence to stainless steel.
Because S. epidermidis is the preeminent cause of orthopaedic prosthetic device infections, it is critically important we gain a thorough understanding of the pathogenesis of these infections and the virulence determinants of this bacterium. Previous studies have defined adherence to the biomaterial as a crucial event in the formation of a S. epidermidis prosthetic device infection.12-14 In addition, biofilm formation appears to be a multi-step process in which PIA plays a pivotal role in the later stages - during cellular aggregation and biofilm maturation.8,13,14 The data from this study suggest PIA also plays a critical role in initial adherence of S. epidermidis to many biomaterials, including those used in orthopaedic prosthetic devices, and it may serve as an important factor in establishment of orthopaedic infections. Additionally, differences in the ability of PIA-positive S. epidermidis 1457 to adhere to various orthopaedic biomaterials suggest certain compounds may be more prone to colonization by PIA-producing S. epidermidis. In certain materials (eg, stainless steel), adherence was especially PIA dependent, whereas in other materials the ability of the organism to produce PIA did not affect the ability of the organism to bind to the biomaterial (eg, titanium). These data are not surprising as staphylococci encode and express multiple adherence factors. It seems logical these adherence factors (including PIA) might have greater affinity for certain bio-materials. By defining which adhesins are important in adherence to orthopaedic biomaterials, it may be possible to formulate prosthetic devices less susceptible to bacterial adherence and hence less inclined to become infected.
Several limitations must be noted to interpret the data. First, in vitro studies have a number of inherent limitations. The pathogenesis of prosthetic device infections is a complex process involving interactions between the pathogen, the biomaterial, and the host. Obviously, in vitro as-says do not account for host defense and other in vivo factors. However, this study provides strong support for further investigation of the importance of S. epidermidis PIA in relevant in vivo models of orthopaedic device infection. Another potential limitation of this in vitro assay is the use of stationary phase organisms. Bacterial cells causing orthopaedic implant infections may be at a different growth phase and may express alternative adhesins or other virulence determinants. Similarly, the biomaterial samples in this study were exposed to a nonphysiological inoculum level. Most likely, orthopaedic implants that become infected are exposed to relatively small numbers of bacteria at the time of implantation, not the very large inoculum used in our in vitro assay.
Our results suggest PIA plays a critical role in initial adherence of S. epidermidis to orthopaedic biomaterials. These findings augment established functions of PIA, which include multicellular aggregation, antibiotic resistance, and immune evasion.6 These data also show S. epidermidis has the differential ability to bind to various orthopaedic biomaterials. Further studies are warranted to assess the importance of PIA in in vivo models of orthopaedic prosthetic infection. It is hoped such investigations may lead to novel therapeutics or improved treatment modalities.
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