Clinical History
A forty-seven-year-old man presented with left elbow pain five years after undergoing open reduction and internal fixation of a comminuted left distal humeral fracture. The patient was a laborer and had continued working with the injured arm despite discomfort. Radiographs demonstrated nonunion of the distal part of the humerus along with hardware failure (Fig. 1, A). The hardware was surgically removed; an intraoperative Gram stain was negative, with fewer than five white blood cells per high-powered field. Given the extent of the tissue inflammation that was observed, it was believed that the safest course would be to remove all hardware, reactive tissue, and devascularized bone. The void left by removal of the bone was filled with a cement spacer impregnated with vancomycin (3.5 g). Final bacterial cultures showed no growth.
One month later, the patient underwent a total elbow arthroplasty with allograft (Fig. 1, B). The immediate postoperative course was uneventful. Eight months later, the patient complained of recurrent left elbow pain. Routine aerobic and anaerobic cultures of an aspirate from the elbow demonstrated no bacterial growth. Due to persistent symptoms, the patient underwent irrigation and débridement and the radial head was resected. Aerobic and anaerobic bacterial cultures again were negative both for the aspirate and tissue samples (no growth).
Thirteen months after the total elbow arthroplasty, the elbow pain recurred. Radiographs showed evidence of loosening about the ulnar component. The humeral component appeared to be well-fixed (Fig. 1, C). An irrigation and débridement was again performed. The implants and cement were removed; a cement spacer that was impregnated with tobramycin (prepared according to the manufacturer's directions) was placed. Final bacterial cultures of samples taken from within the humeral shaft, from the medullary canal of the ulna, and from tissue about the elbow joint again showed no growth. The patient was taken back to the operating room three and one-half months later for removal of the cement spacer. Final bacterial cultures at this point were also negative (no growth).
Five months later, the patient returned with a one-week history of fever and malaise. He had diffuse pain and swelling in the region of the midbrachium. Cloudy fluid was draining from a small sinus track on the anterolateral aspect of the arm. The patient was returned to the operating room for débridement of the wound. Retained cement was removed from the humerus. Intraoperative cultures from aspirated joint fluid at this time did grow methicillin-resistant Staphylococcus aureus. This isolate was found to be susceptible to vancomycin, gentamicin, clindamycin, and sulfamethoxazole-trimethoprim and resistant to nafcillin and erythromycin. Fungal cultures and mycobacterial cultures were all negative (no growth). An eight-week course of intravenous vancomycin and oral rifampin was completed (the patient had previously received multiple other courses of antibiotics empirically as well, usually consisting of intravenous cefazolin followed by oral cephalexin, although ceftriaxone and gatifloxacin had also been briefly administered).
Two years later (at the time of the most recent follow-up), the patient had no evidence of infection. He has a flail elbow resulting in an arm that is only useful when the elbow is supported in a hinged brace.
Confocal Laser Scanning Microscopy and Viability Staining
Tissue and cement recovered during the final revision surgery were collected in sterile tubes and placed on ice. Prior to staining, samples were rinsed by immersion in Ringer solution (one-quarter strength), blotted to remove excess fluid, and then transferred to a 100-mm Petri dish. A dab of Lubriseal grease (Thomas Scientific, Swedesboro, New Jersey) was smeared on the bottom of each Petri dish; the specimen was gently pressed onto the grease, avoiding contact with the center, to immobilize the specimen for microscopic examination. Tissue and cement samples were stained with use of the LIVE/DEAD BacLight kit (Molecular Probes, Eugene, Oregon) by drop-pipetting the manufacturer's recommended concentration directly onto the specimens. Specimens were incubated for fifteen minutes in the dark at room temperature. Excess stain was rinsed away by flooding the plate with phosphate-buffered saline solution and then aspirating. The specimens were submerged in phosphate-buffered saline solution before they were histologically examined with use of a Leica DM RXE upright microscope attached to a TCS SP2 AOBS confocal system (Leica Microsystem, Exton, Pennsylvania). The 488-nm line of the Kr/AG-laser was used as the excitation wavelength and the detector wavelength windows were set such that the "live" stain (SYTO 9; Molecular Probes) appeared green and the "dead" stain (propidium iodide) appeared red. The surface of the bone cement was imaged in reflected confocal mode and made to appear blue in the images. Thus, fresh specimens were examined in their fully hydrated state with minimal preparation.
Nucleic Acid Isolation and Reverse Transcriptase-Polymerase Chain Reaction
Nucleic acid isolations from bacterial reference strains (Staphylococcus aureus American Type Culture Collection [ATCC] 25923 and Staphylococcus epidermidis ATCC 35984) were performed as described7. For the clinical specimens, 200 µL of aspirate fluid obtained from the operative site prior to open surgery was placed in 1 mL of RNAlater (Ambion, Austin, Texas) and stored at -70°C. Cells were pelleted, and 480 µL of hot phenol buffer7 was added. This resuspension then was subjected to phenol-chloroform extraction as previously described7. Recovered nucleic acids, both wound-derived and from the reference bacterial strains noted above, were treated with deoxyribonuclease (DNase) (as per the specifications of the TURBO DNase kit; Ambion) and evaluated for integrity with use of an Agilent bioanalyzer (Model 2100; Agilent, Palo Alto, California), which confirmed little to no degradation. Reverse transcription and subsequent polymerase chain reaction on the recovered ribonucleic acids were then carried out as described7. A set of negative controls to test for contaminating DNA (sterile water was added in place of reverse transcriptase) was also carried out simultaneously. Following reverse transcriptase-polymerase chain reaction, the amplimers were subjected to 1% agarose-gel electrophoresis and visualized with ethidium bromide. The specific primer sequences for Staphylococcus aureus and Staphylococcus epidermidis have been previously described7. In addition, we used a generic staphylococcal primer set (GF-1/GR-2) directed against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene16 and a 23S rRNA Staphylococcus aureus-specific primer set (532720-GGACGACATTAGACGAATCA-532739 and 534038-CGGGCACCTATTTTCTATCT-534019), which were developed and validated in the present study under the same polymerase-chain-reaction-cycling conditions as previously described7.
Confocal Microscopy
At the time of the final operation, confocal laser scanning microscopy of fluid aspirated from the site to be surgically resected revealed the presence of cocci ranging from single cells to large aggregates of grape-like clusters (Fig. 2). The largest clumps were approximately 100 µm in diameter and were estimated to contain many hundreds to thousands of cocci. The bacteria in these clusters stained with SYTO 9 (green) but not with propidium iodide (red), signifying that they were viable.
Examination of tissue associated with the implant revealed similar clusters of viable bacteria (stained green) that were adherent to the tissue (Fig. 3). Host nuclei and fibrous material stained red with propidium iodide. Bacterial clusters protruded from the surface of the host tissue and were similar in appearance to biofilm clusters grown in vitro8,17. Some of the cocci were in the process of division, which was consistent with the results of viability staining. The distribution of biofilm was patchy, with some sites containing large clusters and other regions showing no evidence of infection.
Examination of the residual bone cement revealed both single bacterial coccal cells and clusters of biofilm bacteria attached to the irregular and porous surface (Fig. 4). The clusters were as much as approximately 10 µm in diameter and contained between ten and fifty cells. The distribution of biofilm was patchy, similar to that seen in the tissue samples. Consistent with what was seen in the tissue and aspirate samples, the cocci stained green, indicating viability. The clumps had three-dimensional structure, evidencing an extracellular matrix, and protruded from the surface of the cement. Host cells, visualized by red-stained nuclei, were also seen in close proximity to the cement surface.
Reverse Transcriptase-Polymerase Chain Reaction and Polymerase Chain Reaction
The polymerase chain reaction (reverse transcriptase-polymerase chain reaction) assay for Staphylococcus aureus and Staphylococcus epidermidis in tissue from the elbow showed polymerase-chain-reaction amplimers of the expected molecular weight when a Staphylococcus aureus primer or nonspecific Staphylococcus primers were used on wound material from the patient (as well as on a reference Staphylococcus aureus control strain) (Fig. 5). In contrast, no amplimer was seen when Staphylococcus epidermidis-specific primers were used, signifying that no Staphylococcus epidermidis was present. When no reverse transcriptase enzyme was added, the only reactions that produced amplimer were the non-DNase-treated control reactions. The absence of amplimers from the DNase-treated clinical specimens when reverse transcriptase was omitted, together with the positive reverse transcriptase-polymerase chain reaction results from the DNase-treated clinical specimens, proved that bacterial messenger ribonucleic acid (mRNA) was present.
Note: The authors thank Dr. Luanne Hall-Stoodley and Dr. J. Christopher Post for their assistance with this report.