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

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: 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.