Molecular and Imaging Techniques for Bacterial Biofilms in Joint Arthroplasty Infections

Synovial Fluid Biomarkers for Periprosthetic Infection

“…The recent literature has highlighted the need for improved diagnostics as evidenced by the number of studies attempting to identify the best combination of laboratory tests predicting periprosthetic infection [3,4,9,[14][15][16]. Additionally, new methods of diagnosis such as PCR [12,18], biofilm detection [17,20], and cultures augmented by implant ultrasound [19] have been described and evaluated for diagnostic purposes. The patient, physician, and economic burdens of infected arthroplasties [5,6] are expected to grow rapidly with the increase in joint arthroplasties performed each year.…”

Direct Demonstration of Viable Staphylococcus aureus Biofilms in an Infected Total Joint Arthroplasty

“…Biofilm bacteria are vastly more resistant to conventional antibiotic therapy than are their single planktonic counterparts (unattached solitary bacteria living freely) and are typically difficult to culture by conventional microbiological methods 4,5 . The biofilm paradigm can explain contradictory signs and symptoms that suggest infection but are often associated with negative cultures 6,7 . Moreover, biofilm infections are difficult to detect by simple Gram stain and culture techniques but can persist as a nidus of infection from which recurrent acute exacerbations may arise through episodic planktonic ''showering'' 8 .…”

“…Cells were pelleted, and 480 mL of hot phenol buffer 7 was added. This resuspension then was subjected to phenol-chloroform extraction as previously described 7 . 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.…”

“…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 described 7 . 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 described 7 . In addition, we used a generic staphylococcal primer set (GF-1/GR-2) directed against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene 16 and a 23S rRNA Staphylococcus aureus-specific primer set (532720-GGACGA-CATTAGACGAATCA-532739 and 534038-CGGGCACCTATT-TTCTATCT-534019), which were developed and validated in the present study under the same polymerase-chain-reactioncycling conditions as previously described 7 .…”