Protocols for processing of tissue from arthroplasty infections vary and might affect the recovery of bacteria. We compared homogenization, bead beating and enzymatic disruption for recovery of live bacteria from tissue samples.

Suspensions of Staphylococcus aureus and Escherichia coli were prepared as controls. Three samples were taken from each and the first was bead beaten, the second homogenized, and Proteinase K was added for 10 and 30 minutes to the third sample before culturing. In addition, artificially inoculated pork tissue and known infected human tissue samples were processed by either homogenization or bead beating prior to cultures and results were compared.

Number of cycles of bead beating and homogenization and duration of Proteinase K treatment had significant effects. Bead beating for 2 and 4 cycles reduced the yield of S.aureus to 52% and 20% of control, and E.coli to 33% and 8%. Homogenization for 2 and 4 cycles reduced S.aureus to 86% and 65% of control, and E.coli to 90% and 87%. Proteinase K for 10 minutes and 30 minutes reduced the yield of S.aureus to 75% and 33% of control, and E.coli to 91% and 49% respectively. Inoculated Pork tissue showed a reduction in S.aureus recovery of 90% for bead beating compared to homogenization, and 80% in the case of E.coli. Bead beating of infected human tissue samples reduced the yield by 58% compared to homogenization.

Bead-beating is a common recommended method of processing tissue from arthroplasty cases. However, even though it produces a homogeneous sample, it does so at the cost of significant loss of viable bacteria. Homogenization and 10 minutes of Proteinase K incubation are almost equivalent, but the homogenizer is preferred being more controllable and cheaper. This should help to define guidelines for diagnosing infections using tissue samples.

Metal instrumentation (rods and screws) is used to stabilise the spine after trauma, malignancy or deformity. Approx 3% become infected often necessitating removal of metal. At surgery tissue samples and metal are removed for culture, but many clinical laboratories are not equipped to process metal or use simple culture methods. The causative bacteria exist as biofilms on the metal and they are often anaerobic and slow-growing, so conventional culture methods often fail to detect them. Also, they are common contaminants leading to diagnostic uncertainty. We have established a laboratory protocol to overcome these problems.

Removed metalwork was sonicated and the sonicate centrifuged and the supernatant discarded. Quantitative aerobic and anaerobic culture of the resuspended pellet for 14 days and microscopy were carried out.

Metalwork from 11 suspected infected cases was culture-positive (median 2857, 60–5000cfu/mL). Microscopy revealed an infection due to Candida albicans that would not have been detected otherwise. Bacteria were isolated from 8 of 10 non-infected cases (median 15, 0–35 cfu/mL). Conventionally processed samples failed to grow in 4 infected cases. (cfu/mL infected vs noninfected cases p=0.0093)

Micro-organisms on spinal metalwork grow as biofilms and they require sonication to dislodge them. The causative bacteria are slow-growing and P acnes is anaerobic and requires prolonged incubation. S epidermidis and P acnes are common contaminants and quantitative culture helps to distinguish pathogens from contaminants, removing the diagnostic uncertainty that conventional methods give. Microscopy of the sonicate can reveal micro-organisms that fail to grow on culture. We recommend that sonication of metalwork, prolonged anaerobic incubation and quantitative culture be adopted to improve diagnostic clarity for spinal instrumentation infections.

Aim

To test the hypothesis that surface skin swabs taken after skin preparation with alcoholic povidone iodine (APVPI) would not grow bacteria, whereas full thickness biopsies taken from the line of surgical incision would grow bacteria.

Background

Antibiotic loaded bone cement spacers are used as an adjunct to treatment in 2-stage arthroplasty revisions. If release of the correct choice of antimicrobials is optimised, systemic therapy might be curtailed and emergence of resistance minimised. Aims: To determine the elution period of antimicrobials from bone cement with and without a copolymer, polyvinylpyrrolidone (PVP) and to limit resistance development by the use of two or more antimicrobials.

Background

Deep infection rates of 1 - 2% following primary hip and knee arthroplasty are mainly due to endogenous contamination of the surgical site from bacteria within the patient's own skin. However surgical skin preparation removes only bacteria from the surface of the skin, leaving viable bacteria in the deeper layers of the skin within hair follicles and sweat and sebaceous glands. The aim of our study was to test the hypothesis that surface skin swabs taken after skin preparation with alcoholic povidone iodine would not grow bacteria, whereas full thickness biopsies taken from the line of surgical incision would grow bacteria.

Introduction

Open fractures occur with an annual incidence of 11.5 per 100,000 (6900 pa in UK). Infection rates, even with intravenous broad-spectrum antibiotics, remain as high as 22%. For this reason necessary bone grafting is usually delayed until soft-tissue cover of the bone injury is achieved. A biodegradable bone graft that released sustained high concentrations of antibiotics and encouraged osteogenesis, that could be implanted safely on the day of injury would reduce infection rates and avoid reoperation and secondary grafting. The non –union rate (approx 350 pa in UK) should also be reduced. Such a graft, consisting of a PLA/PGA co –polymer and containing antibiotics, is under development and here we report assessment of spectrum and duration of antimicrobial activity and effect of addition of antibiotics on mechanical properties.

Prosthetic joint infection (PJI) is an increasing problem and management commonly involves prosthesis removal with serious consequences. Biofilm-forming staphylococci are the most common causative organisms with Staphylococcus aureus being most virulent and methicillin-resistant Staphylococcus aureus (MRSA) more than doubling the infection mortality rate. Bacterial adhesion is an essential primary event in biofilm formation and infection establishment. The development of a novel combination vaccine programme to prevent staphylococcal PJI by directing antibody against factors involved in adhesion and biofilm formation, and investigation of S. aureus binding-domains as potential vaccine components for adhesion inhibition is described.

Selected target antigens included the S. aureus fibronectin-binding protein (FnBP) and iron-regulated surface determinant (IsdA), which have been shown to be important for infection establishment and predominantly bind to host fibronectin and fibrinogen respectively. Escherichia coli clones harbouring recombinant S. aureus binding-domain DNA sequences were used for expression and purification of antigen domains. In vitro antibody evaluation determined whether immune inhibition of bacteria - ligand binding can significantly impact on attachment to plasma-conditioned biomaterial (in presence of other bacterial ligands).

Adhesion of homologous and heterologous (MRSA PJI isolate) S. aureus to plasma-conditioned steel was significantly reduced (approximately 50 percent average reduction, p < 0.0001) when pre-exposed to anti-rFnBP-A antiserum (with pre-immune serum control) that was 50-fold more dilute than the actual titre from immunisation. Inhibition was related to ligand presence but not staphylococcal Protein A, and reduced adhesion was not observed with the mutant strain, indicating specific inhibitory antibody involvement, and demonstrating for the first time the potential of rFnBP-A for prevention of S. aureus PJI. Adhesion-inhibitory activity was also observed with a purified IgG-fraction of rIsdA antiserum but this activity appeared to be masked by non-IsdA - related interactions when non-IgG - purified antiserum was assessed.

Background: Prosthetic joint infection (PJI) is most commonly caused by skin-derived, biofilm-forming staphylococci, with Staphylococcus aureus being most virulent and MRSA becoming a substantial problem. Cephaloporins are almost universally used as prophylaxis against PJI, yet Methicillin - resistant S aureus (MRSA) is becoming increasingly common in hospitals, nursing homes and now in the community. Such strains are not susceptible to cephalorsporins or to a range of other antimicrobials. In view of this increasing antibiotic resistance, an alternative approach to preventing S. aureus PJI is needed, and we propose that vaccination is a promising approach. Having regard to the distinct pathogenesis of PJI, this must target key events in infection establishment, such as adhesion to the implant, via the plasma conditioning film, mediated by bacterial binding proteins. It must also have the potential to protect against all S. aureus regardless of antibiotic resistance profile. Fibronectin-binding protein-A (FnBP-A) is one example, but the potential of FnBP-A as a PJI vaccine candidate has not been thoroughly investigated and data in previous literature are contradictory.

Methods: Here, polyclonal rabbit antibody against recombinant(r) FnBP-A binding domain was produced and investigated for the first time for activity against S. aureus adhesion to rabbit plasma-conditioned steel coupons in-vitro.

Results: The adhesion of homologous S. aureus 8325-4 (fnbA+, fnbB+), and a heterologous MRSA arthroplasty isolate was significantly (p < 0.05) reduced when pre-exposed to anti-FnBP-A antiserum (un-purified and IgG-purified), compared to pre-exposure with pre-immune serum. This was not observed with mutant strain S. aureus DU5883 (fnbA?, fnbB?), indicating the involvement of FnBP-A – specific inhibitory antibody (IgG). Results clearly demonstrate the potential of rFnBP-A binding domain as a vaccine antigen for prevention of PJI and merit further investigation.

The implications of this are that vaccination using this peptide might be expected to protect patients about to undergo elective arthroplasty from infection with S aureus whatever its antibiotic susceptibility, so offering a realistic solution to the problem of increasing resistance.

Introduction: Propionibacterium acnes (P. acnes), a common anaerobic skin commensal, has been implicated in biomaterial-related infections (BRI). Bacteria can adhere to biomaterial surfaces and grow as a bio-film held together by exopolymer, exhibiting increased antimicrobial resistance. To our knowledge, images of P. acnes biofilms have not previously been published. We have demonstrated the ability of P. acnes to adhere to surgical steel and to develop a biofilm on this material. However its ability to adhere to and develop a biofilm on titanium, a commonly used surgical implant material, has not been fully investigated.

Aims:

To determine the quantitative adherence and biofilm development of P. acnes on titanium compared to surgical steel.

Method: Six clinical isolates of P. acnes were assayed for adherence to materials with and without plasma glycoprotein conditioning film by chemiluminescence and culture. Biofilm development was assessed by chemiluminescence, fluorescence microscopy, environmental (ESEM) and scanning electron microscopy (SEM). Mature biofilms were exposed to plasma concentrations of penicillin and quantified by chemiluminescence and culture. Unpaired student’s t tests and univariate linear regression models were calculated using SPSS software (version 12).

Results: Univariate linear regression showed that P. acnes adherence to titanium was 18% (p=0.001) greater than to steel. Adherence was reduced by the presence of the conditioning film on titanium by 28% (p=0.001), but this made no significant difference to P. acnes adherence to steel. P. acnes biofilms were clearly demonstrated, along with bacterial expolymer, showing an interesting similarity to biofilms of S. epidermidis. P. acnes grows as a thick biofilm on both materials held together by exopolymer and our preliminary results suggest that biofilms on titanium might be less susceptible to antimicrobials after 24 hours of penicillin treatment; a reduction of 94% on steel and 81% on titanium (p=0.057, p=0.39 resp).

Conclusions: P. acnes adheres to steel and titanium, a crucial first step in BRI. Greater numbers of P. acnes adhere to titanium than to steel. The naked surface of titanium is microporous, assisting adhesion. A conditioning film reduces P. acnes adherence to titanium but not to steel. P. acnes develops as a biofilm on steel and titanium. Results indicate that pathogenesis of P. acnes infection on titanium is more successful than on steel. P. acnes biofilms on titanium may be harder to eradicate with antimicrobial agents.

Introduction: One of the most important mechanisms S. epidermidis uses to establish infection on biomaterials is biofilm formation, in which adhesion and the production of polysaccharide intercellular adhesin (PIA) are key factors. Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to inhibit S. epidermidis biofilm formation and may be useful in prevention or treatment of implant infections (1,2). The potential of these drugs was evaluated by determining the effects of the NSAIDs on bacterial growth, adhesion to bare and conditioned polymethyl-methacrylate (PMMA), on biofilm development, and on established biofilms.

Methods: A PIA-deficient mutant and wild type strain (gift of Prof. D. Mack, Hamburg) and 3 clinical isolates of S. epidermidis were used. The NSAIDs were salicylic acid, acetylsalicylic acid, ibuprofen and phenylbutazone. Their effects on bacterial growth rate and viability were assessed. For adhesion assay, bacteria were exposed to a 1mM concentration of each drug and allowed to adhere for 1h to bare or human plasma – conditioned PMMA before being sonicated and quantified by chemiluminescence and culture. For biofilm assays, bacteria were grown on silicone discs in the presence of various drug concentrations for 4 days before being sonicated and quantified as above. Mature (4 day) biofilms were also exposed to the drugs for a further 4 days and quantified similarly, to assess the effect on established biofilms.

Results: All NSAIDs tested significantly (P< 0.05) reduced the growth rate and viability of each strain, in a concentration – dependent manner. Reduction of adhesion was observed on bare PMMA suggesting interference with either vitronectin – binding protein or charge / hydrophobic interactions. This was independent of the effect on growth. However, adhesion to plasma – conditioned PMMA, presumably mediated by MSCRAMMs, was not significantly affected. Reduction of biofilm formation was observed for all strains and was concentration – dependent, suggesting that inhibition of PIA synthesis was not responsible. There was a significant effect on established biofilms, this was also concentration dependent.

Conclusions:

All four NSAIDs reduced S. epidermidis growth rate and viability, but at concentrations above those achievable therapeutically.

Objective: To compare the in-vitro antimicrobial action of surgical irrigation fluids: 0.9% saline, 2g/L cephradine, 80mg/L gentamicin, 10% povidone-iodine (PVP-I) and 40ppm aqueous iodine (laq) for activity against Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli in the presence of blood, plasma and saline.

Materials and Methods: 5mL of antimicrobial agent was added to 5ml of blood, plasma or saline containing 10^5–10⁶ CFU/mL of the test bacterium. At 15 seconds, 1 minute, 5 minutes, 1 hour and 2 hours, a 1mL sample was inactivated in 9mL of 0.5% sodium thiosulphate. The bacterial numbers were determined using a biochemical assay (Chemiluminescence) with a calibration curve and by spread plate counts. The data were transformed by a logarithmic function and analysed by linear regression to give 95% confidence intervals for their gradient of change over 2 hours. Significant differences were defined at the 5% level.

Results: In saline. All bacteria were killed within 15 seconds with PVP-I and Iaq, but showed no significant reduction with saline, cephradine or gentamicin. In plasma. E. coli was killed within 15 seconds with all irrigation fluids. S. aureus and S. epidermidis showed no significant reduction with saline, Iaq or cephradine, but did show a significant reduction in the presence of gentamicin. With PVP-I, all S. epidermidis were killed within 15 seconds and all S. aureus within 5 minutes. In blood. E.coli with PVP-I, Iaq and cephradine showed no significant reduction. E. coli with gentamicin did show a significant reduction. Both staphylococci showed a significant reduction over 2 hours with PVP-I and gentamicin, but no significant reduction with saline, cephradine and Iaq.

Conclusions: As blood has a strong chemical inactivating effect on iodine – based formulations, Iaq and PVP-I cannot be recommended for surgical irrigation. Gentamicin should be used in preference to cephradine in surgical irrigation fluids if an antimicrobial agent is required. The residual immunological components (particularly complement) in blood and plasma may enhance the susceptibility of bacteria to antimicrobial agents.

Introduction and Aims: A phenotypic and proteomic approach has identified novel targets for the development of a DNA vaccine to prevent Staphylococcus aureus infection in orthopaedics.

Approximately 1% of joint replacement operations are complicated by infection. Thirty percent of these infections are due to S.aureus, which is often difficult to treat because of antibiotic resistance. As treatment of these infections is challenging, prevention with a vaccine is a very attractive option.

Method: To infect a joint replacement, bacteria must first adhere to its surface. This adherence is mediated by specific adhesion proteins; the expression of which is controlled by virulence regulator genes within the bacterial cell. A DNA vaccine is being developed which targets this regulatory apparatus, thus preventing bacterial adhesion, allowing the immune system to rapidly clear any potential S.aureus infection.

Results: Mutations of the agr,sar and sae virulence regulator genes have been made. Their properties have been explored using a flow cell system, which uses a scanning confocal laser microscope and image analysis software to accurately provide quantitative data in real-time of biofilm formation. We have shown that the sae mutant does not form biofilm in the same was as wild-type S.aureus. We have also shown that it does not adhere to steel as well as its wild-type counterpart.

Conclusion: For such a dramatic difference in biofilm forming properties to be evident, there must be a difference in the adhesion proteins produced by the wild-type and the mutant bacteria. Gel-electrophoresis has compared protein expression of sae mutant and wild-type bacteria and identified differences. Those proteins which are not expressed in the non-biofilm-forming mutant are sequenced and from the protein sequences, DNA sequences are identified that will form part of the candidate DNA vaccine.

Objective: To determine whether Propionibacterium acnes is able to adhere to implant materials, and to develop biofilms.

Background: Most orthopaedic implant infections are caused by staphylococci, which express adhesins and can adhere to biomaterials and to plasma glycoprotein conditioning films. They then produce exopolymers and develop biofioms. P acnes, being anaerobic, is often missed as a cause of implant infection and might be more common than is realised, yet little is known about its virulence factors (ability to adhere to biomaterials or conditioning film and biofilm development).

Materials & Methods: Surgical steel or silicone coupons, with and without plasma conditioning film, were exposed to three clinical isolates of P acnes and examined by cultural methods and chemiluminescence for adherence. In further experiments, the coupons were incubated anaerobically with the P acnes strains for several days. They were then rinsed, fixed and processed for scanning electron microscopy (SEM). In a third set of experiments, coupons were again incubated anaerobically with P acnes and examined by laser confocal microscopy (LCM) for biofilm development.

Results: All three isolates of P acnes were able to adhere to the biomaterials, to a degree similar to that of a clinical isolate of Staphylococcus aureus, though not as strongly as Staphylococcus epidermidis. Unlike with the staphylococci, the presence of a conditioning film did not make a significant difference. SEM and LCM revealed biofilm development morphologically similar to that seen with S epidermidis. Exopolymer production was also demonstrated.

Conclusions: P acnes is able to adhere to biomaterials but not so avidly as S epidermidis. The adherence is not enhanced by plasma conditioning film. However, once adhered, P acnes is capable of developing a biofilm morphologically indistinguishable from that of S epidermidis. This probably explains the role of P acnes in implant infection, and the therapeutic difficulty it often poses.

Infection of orthopaedic implants is a significant problem, with increased antibiotic resistance of adherent ‘biofilm’ bacteria causing difficulties in treatment. We have investigated the in vitro effect of a pulsed electromagnetic field (PEMF) on the efficacy of antibiotics in the treatment of infection of implants.

Five-day biofilms of Staphylococcus epidermidis were grown on the tips of stainless-steel pegs. They were exposed for 12 hours to varying concentrations of gentamicin or vancomycin in microtitre trays at 37°C and 5% CO₂. The test group were exposed to a PEMF. The control tray was not exposed to a PEMF. After exposure to antibiotic the pegs were incubated overnight, before standard plating onto blood agar for colony counting.

Exposure to a PEMF increased the effectiveness of gentamicin against the five-day biofilms of Staphylococcus epidermidis. In three of five experiments there was reduction of at least 50% in the minimum biofilm inhibitory concentration. In a fourth experiment there was a two-log difference in colony count at 160 mg/l of gentamicin. Analysis of variance (ANOVA) confirmed an effect by a PEMF on the efficacy of gentamicin which was significant at p < 0.05. There was no significant effect with vancomycin.

Most infections in arthroplasty are caused by staphylococci, about half being due to S. aureus. One of the most worrying aspects of this organism, and particularly of MRSA, is increasing multiple drug resistance, so that antimicrobial prophylaxis is probably already compromised. Vaccination offers a novel approach to overcome this. Detailed consideration of the pathogenesis of prosthesis–related infection indicates that a) prosthetic material rapidly becomes coated after implantation with plasma–derived conditioning film, and b) attachment of the bacteria to the conditioning film, by means of specific bacterial surface binding proteins, is an essential primary event. We hypothesise that antibodies to these binding proteins will block bacterial adhesion to the prosthesis, so reducing the incidence of infection. The aim of this research was to determine the effect of specific antibodies to two binding proteins (fibronectin - and fibrinogen–binding proteins, Fnbp and Fgbp respectively) on bacterial adherence to orthopaedic biomaterials coated with plasma conditioning film.

Antibodies to recombinant sequences of Fnbp and Fgbp were raised in rabbits. A strain of S. aureus bearing a genetically inserted fluorescent reporter (GFP) was used. Orthopaedic biomaterials (steel, titanium and PMMA) were coated with FFP–derived conditioning film, placed in a specially–designed flow cell and exposed to a flow of S. aureus for 3h. Images were captured every 15min and analysed for adherent bacteria using image analysis software. The experiment was repeated in the presence of the antibodies and the results compared.

Each antibody reduced the number of bacteria binding to all three materials by greater than 50%. Combining the two antibodies gave similar results to those when they were used individually.

These preliminary results suggest that while further research is required, vaccination aimed at blocking bacterial attachment to conditioning film on implanted prostheses might reduce the incidence of S. aureus infection in arthroplasty. If so, this would apply even to MRSA. Questions remaining to be addressed include the clinical relevance of a 50% reduction in attachment, and future research will attempt to link this to a reduction in infection.

The release of gentamicin sulphate, sodium fusidate and diethanolamine fusidate from Palacos and CMW cements was studied using elution and serial plate transfer tests. Further tests were made to assay the drug remaining in the cement after antibacterial activity could no longer be detected by the above methods, to detect the sustained slow release of the residual drug, and to ascertain the mechanism of release. The results confirmed that the release of gentamicin sulphate could be detected for longer from Palacos cement than from CMW cement, but the opposite was true for sodium fusidate. Little difference was found in the case of diethanolamine fusidate. Comparison of elution and serial plate transfer tests, and of results of elution in buffers of different pH, demonstrated that the test method employed had a significant effect on the results, and the omission of details of methodology from some publications made comparison and evaluation of results difficult. Varying quantities of residual drug were found in cement from which antibacterial activity could no longer be demonstrated; further tests for sustained, slow release showed that the antibiotic did not remain fixed in the cement but was released at a rate too slow to be detected in the elution and serial plate transfer tests. It is concluded that antibiotics are released from the cement by a process of diffusion, but tests to determine the mechanism of diffusion were unhelpful. The theory of diffusion of drugs through solid matrices, and the clinical implications of the experimental findings, are discussed.