Staphylococcus aureus Aggregates on Orthopedic Materials under Varying Levels of Shear Stress.

Periprosthetic joint infection (PJI) occurring after artificial joint replacement is a major clinical issue requiring multiple surgeries and antibiotic interventions. Staphylococcus aureus is the bacterium most commonly responsible for PJI. Recent in vitro research has shown that staphylococcal strains rapidly form aggregates in the presence of synovial fluid (SF). We hypothesize that these aggregates provide early protection to bacteria entering the wound site, allowing them time to attach to the implant surface, leading to biofilm formation. Thus, understanding the attachment kinetics of these aggregates is critical in understanding their adhesion to various biomaterial surfaces. In this study, the number, size, and surface area coverage of aggregates as well as of single cells of S. aureus were quantified under various conditions on different orthopedic materials relevant to orthopedic surgery: stainless steel (316L), titanium (Ti), hydroxyapatite (HA), and polyethylene (PE). It was observed that, regardless of the material type, SF-induced aggregation resulted in reduced aggregate surface attachment and greater aggregate size than the single-cell populations under various shear stresses. Additionally, the surface area coverage of bacterial aggregates on PE was relatively high compared to that on other materials, which could potentially be due to the rougher surface of PE. Furthermore, increasing shear stress to 78 mPa decreased aggregate attachment to Ti and HA while increasing the aggregates' average size. Therefore, this study demonstrates that SF induced inhibition of aggregate attachment to all materials, suggesting that biofilm formation is initiated by lodging of aggregates on the surface features of implants and host tissues.IMPORTANCE Periprosthetic joint infection occurring after artificial joint replacement is a major clinical issue that require repeated surgeries and antibiotic interventions. Unfortunately, 26% of patients die within 5 years of developing these infections. Staphylococcus aureus is the bacterium most commonly responsible for this problem and can form biofilms to provide protection from antibiotics as well as the immune system. Although biofilms are evident on the infected implants, it is unclear how these are attached to the surface in the first place. Recent in vitro investigations have shown that staphylococcal strains rapidly form aggregates in the presence of synovial fluid and provide protection to bacteria, thus allowing them time to attach to the implant surface, leading to biofilm formation. In this study, we investigated the attachment kinetics of Staphylococcus aureus aggregates on different orthopedic materials. The information presented in this article will be useful in surgical management and implant design.

Pilot study: Internally cooled orthopedic drills - standard sterilization is not enough?

Bone drilling causes focal temperature rise due to metal-to-bone contact, which may result in thermal osteonecrosis. Newly constructed internally cooled medical drill of an open type decreases temperature rise at a point of metal-to-bone contact although standard sterilization of such a drill could be inadequate due to bacteria retention within the drill lumen. The aim of this pilot study was to examine the effectiveness of sterilization and to propose sterilization recommendations for internally cooled open type bone drills. Unused internally cooled medical steel bone drills were tested. Drills were contaminated with Pseudomonas aeruginosa, Bacillus sp., beta-hemolytic Streptococcus sp., Enterobacter sp. and methicillin-resistant Staphylococcus pseudintermedius and then incubated for 24 hours at 37 °C. Afterwards, drills were autoclaved for 15, 20 and 30 minutes at 132 °C and 2.6 bar. When 15-minute sterilization was used, one out of 16 drills was contaminated with Pseudomonas aeruginosa, while the other 15 drills were sterile. Extended cycle sterilization in autoclave lasting for 20 and 30 minutes resulted in 100% sterility of all drills tested. In conclusion, lumened drills should be exposed to extended sterilization times in autoclave. Minimal recommended time for sterilization of lumened drills is 20 minutes.

[Use of scanning electron microscopy to study Candida albicans biofilms on the surface of base plastics of removable orthopedic constructions.]

The most common pathology in the clinic of orthopedic dentistry is the presence of partial adentia in patients, manifested in the form of defects of dentition of various localization and length. Removable orthopedic structures in the oral cavity are a potential place for adhesion and colonization of microorganisms. The aim of the research was to study Candida albicans biofilms on the surface of base plastics of removable orthopedic structures using scanning electron microscopy. 175 cultures of C. albicans were isolated and identified from the oral mucosa of patients at various stages of orthopedic rehabilitation. When studying the surface of samples of plastics of hot and cold type polymerization and Candida biofilms using a JEOL JCM 5700 scanning electron microscope (JEOL, Japan), features of biofilm formation were established. An assessment of the nature of the manifestation of the hemagglutinating activity of clinical strains of Candida fungi in the hemagglutination test with human erythrocytes I (O), II (A) of the human and guinea pig blood groups was carried out. The total number of hemagglutinating strains was 37.14%, with the prevalence of the proportion of manna-resistant (MRHA) cultures - 23.43% of cases. Micrographs of the C. albicans yeast-like biofilm biofilm were obtained on the surface of hot and cold-type plastics in incubation dynamics. Scanning electron microscopy revealed the most pronounced changes in the surface of hot plastics of polymerization compared to cold plastics with long incubation of C. albicans, which characterize the loosening of plastics and the appearance of cracks on the surface, and the cracking of a yeast-like fungus biofilm was noted.

Ornamental morphology of ionic liquid functionalized ternary doped N, P, F and N, B, F-reduced graphene oxide and their prevention activities of bacterial biofilm-associated with orthopedic implantation.

The multifunctional biological active material design for bone tissue engineering is essential to induce osteoblast cell proliferation and attachment. Adhesion of bacteria on biomaterials to produce biofilms can be major contributors to the pathogenesis of implant material associated infections. This research work focuses on NPF& NBF elemental doping and functionalization of reduced graphene oxide using an imidazolium-based ionic liquid such as BMIM PF6 and BMIM BF4 by hydrothermal method. The resulting tri doped reduced graphene oxide (NPF-rGO and NBF-rGO) composite was further used as a scaffold for bone tissue engineering and anti-biofilm activities. The observation of the effect of NPF-rGO and NBF-rGO on the morphology, adhesion and cell proliferation of HOS cell was investigated. Moreover, the tri doped composite tested its antibiofilm properties against B. subtilis, E. coli, K. pneumoniae, and P. aeruginosa pathogenic bacteria. In-vitro studies clearly show the effectiveness of N, P, B, and F doping promoting the rGO mineralization, biocompatibility, and destruction of bacterial biofilm formation. The result of this study suggests that NPF-rGO and NBF-rGO hybrid material will be a promising scaffold for bone reaeration and implantation with a minimal bacterial infection.

Orthopedic implant-associated infections caused by Cutibacterium spp. - A remaining diagnostic challenge.

BACKGROUND: The definition criteria and clinical characteristics of implant-associated infection (IAI) caused by Cutibacterium (formerly Propionibacterium) spp. are poorly known. We analyzed microbiologically proven Cutibacterium orthopedic IAI in a prospective cohort. METHODS: Patients with periprosthetic joint infections (PJI) and fixation device-associated infections (FDAI) caused by Cutibacterium spp. were prospectively included. IAI was defined by significant growth of Cutibacterium spp. and presence of at least one non-microbiological criterion for infection. The McNemar's chi-squared or binomial test was used to compare the performance of diagnostic tests. RESULTS: Of 121 patients with Cutibacterium IAI, 62 patients (51%) had PJI and 59 (49%) had FDAI. 109 infections (90%) were caused by C. acnes and 12 (10%) by C. avidum. The median time from implantation until diagnosis of infection was 15.7 months (interquartile range, 5-46.5 months). Clinical local signs were present in 30 patients (28%) and radiological implant loosening in 64 patients (63%). Culture sensitivity of sonication fluid was 84%, of peri-implant tissue 84% and of synovial or peri-implant fluid 56% after 14 days of incubation. CONCLUSION: Cutibacterium IAI was diagnosed late in the disease course and presented with subtle signs. Prolonged culture incubation and implant sonication improved the poor performance of conventional microbiological tests. Due to lack of reliable diagnostic tests, Cutibacterium remains difficult to detect making the diagnosis challenging.

Effects of titanium nanotubes on the osseointegration, cell differentiation, mineralisation and antibacterial properties of orthopaedic implant surfaces.

The development and pre-clinical evaluation of nano-texturised, biomimetic, surfaces of titanium (Ti) implants treated with titanium dioxide (TiO2) nanotube arrays is reviewed. In vitro and in vivo evaluations show that TiO2 nanotubes on Ti surfaces positively affect the osseointegration, cell differentiation, mineralisation, and anti-microbial properties. This surface treatment can be superimposed onto existing macro and micro porous Ti implants creating a surface texture that also interacts with cells at the nano level. Histology and mechanical pull-out testing of specimens in rabbits indicate that TiO2 nanotubes improves bone bonding nine-fold (p = 0.008). The rate of mineralisation associated with TiO2 nanotube surfaces is about three times that of non-treated Ti surfaces. In addition to improved osseointegration properties, TiO2 nanotubes reduce the initial adhesion and colonisation of Staphylococcus epidermidis Collectively, the properties of Ti implant surfaces enhanced with TiO2 nanotubes show great promise. Cite this article: Bone Joint J 2018;100-B(1 Supple A):9-16.

Comparative Genomics Study of Staphylococcus epidermidis Isolates from Orthopedic-Device-Related Infections Correlated with Patient Outcome.

Staphylococcus epidermidis has emerged as an important opportunistic pathogen causing orthopedic-device-related infections (ODRI). This study investigated the association of genome variation and phenotypic features of the infecting S. epidermidis isolate with the clinical outcome for the infected patient. S. epidermidis isolates were collected from 104 patients with ODRI. Their clinical outcomes were evaluated, after an average of 26 months, as either "cured" or "not cured." The isolates were tested for antibiotic susceptibility and biofilm formation. Whole-genome sequencing was performed on all isolates, and genomic variation was related to features associated with "cured" and "not cured." Strong biofilm formation and aminoglycoside resistance were associated with a "not-cured" outcome (P = 0.031 and P < 0.001, respectively). Based on gene-by-gene analysis, some accessory genes were more prevalent in isolates from the "not-cured" group. These included the biofilm-associated bhp gene, the antiseptic resistance qacA gene, the cassette chromosome recombinase-encoding genes ccrA and ccrB, and the IS256-like transposase gene. This study identifies biofilm formation and antibiotic resistance as associated with poor outcome in S. epidermidis ODRI. Whole-genome sequencing identified specific genes associated with a "not-cured" outcome that should be validated in future studies. (The study has been registered at ClinicalTrials.gov with identifier NCT02640937.).

In Vivo Assessment of Phage and Linezolid Based Implant Coatings for Treatment of Methicillin Resistant S. aureus (MRSA) Mediated Orthopaedic Device Related Infections.

Staphylococcus comprises up to two-thirds of all pathogens in orthopaedic implant infections with two species respectively Staphylococcus aureus and Staphylococcus epidermidis, being the predominate etiological agents isolated. Further, with the emergence of methicillin-resistant S. aureus (MRSA), treatment of S. aureus implant infections has become more difficult, thus representing a devastating complication. Use of local delivery system consisting of S.aureus specific phage along with linezolid (incorporated in biopolymer) allowing gradual release of the two agents at the implant site represents a new, still unexplored treatment option (against orthopaedic implant infections) that has been studied in an animal model of prosthetic joint infection. Naked wire, hydroxypropyl methylcellulose (HPMC) coated wire and phage and /or linezolid coated K-wire were surgically implanted into the intra-medullary canal of mouse femur bone of respective groups followed by inoculation of S.aureus ATCC 43300(MRSA). Mice implanted with K-wire coated with both the agents i.e phage as well as linezolid (dual coated wires) showed maximum reduction in bacterial adherence, associated inflammation of the joint as well as faster resumption of locomotion and motor function of the limb. Also, all the coating treatments showed no emergence of resistant mutants. Use of dual coated implants incorporating lytic phage (capable of self-multiplication) as well as linezolid presents an attractive and aggressive early approach in preventing as well as treating implant associated infections caused by methicillin resistant S. aureus strains as assessed in a murine model of experimental joint infection.

Role of Sonication for Detection of Infection in Explanted Orthopaedic Trauma Implants.

OBJECTIVES: Sonication is a new technology that uses high-frequency sound waves to mechanically dislodge bacteria adherent in biofilms. Unlike arthroplasty, its role in orthopaedic trauma has not been described. The goal of this study was to explore the utility of sonication in orthopaedic trauma. DESIGN: Retrospective review. SETTING: Level I trauma center. PATIENTS: One hundred forty-six sonicated metallic orthopaedic devices from September 2010 to May 2013 were included. Patients were divided into 3 groups: clinically infected, elective implant removals, and nonunion. INTERVENTION: Sonication culture results were retrospectively reviewed for all patients undergoing implant removal. OUTCOMES: Sonication results were the primary study outcome and were considered positive for culture growth if equal to or greater than 20 colony-forming units per plate. RESULTS: In 32 patients with clinical infection, tissue cultures were positive in 30 (94%) and negative in 2 (6%). In contrast, sonication cultures were positive in 19 patients (59%) and did not identify additional organisms. Of the 72 patients who underwent elective implant removal, 52 had pain. Sonication cultures were positive in 5 of these 52 patients (10%) and in 0 of 20 patients with no pain. Sonication culture results were negative in all 42 patients who underwent nonunion surgery. CONCLUSIONS: Sonication of orthopaedic trauma implants in patients with clinically apparent infection or "aseptic" nonunion offered negligible additional information. Sonication demonstrated a positive microbiologic yield in a subset of patients with painful implants; further research is required to better establish the frequency of subclinical infection and to determine the diagnostic role of traditional cultures and sonication. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Microbial colonisation of orthopaedic tourniquets: a potential risk for surgical site infection.

Pneumatic tourniquets have been used in orthopaedic surgery to get avascular fields. Sixteen such tourniquets were analysed for microbial colonisation. Samples were taken from two inner and two outer areas of each tourniquet and cultured on sheep blood agar. Eight of these were wiped with Savlon and the rest with Sterillium solution. Post-treatment samples from the same sites were again cultured. After incubation, colonies from each site were identified and counted. It was observed that the tourniquets were colonised with coagulase-negative staphylococci, Staphylococcus aureus, Bacillus, diphtheroids, Pseudomonas, Acinetobacter, enterococci, enterobacteria, and Candida. On treating with Savlon and Sterillium, there was 92.18% and 95.70% reduction in the colony count, respectively.

Imaging bacteria and biofilms on hardware and periprosthetic tissue in orthopedic infections.

Infection is a major complication of total joint arthroplasty (TJA) surgery, and even though it is now as low as 1 % in some hospitals, the increasing number of primary surgeries translates to tens of thousands of revisions due to prosthetic joint infection (PJI). In many cases the only solution is revision surgery in which the hardware is removed. This process is extremely long and painful for patients and is a considerable financial burden for the health-care system. A significant proportion of the difficulties in diagnosis and treatment of PJI are associated with biofilm formation where bacteria attach to the surface of the prosthesis and periprosthetic tissue and build a 3-D biofilm community encased in an extracellular polymeric slime (EPS) matrix. Bacteria in biofilms have a low metabolic rate which is thought to be a major contributor to their recalcitrance to antibiotic treatment. The diagnosis of biofilm infections is difficult due to the fact that bacteria in biofilms are not readily cultured with standard clinical microbiology techniques. To identify and visualize in situ biofilm bacteria in orthopedic samples, we have developed protocols for the collection of samples in the operating room, for molecular fluorescent staining with 16S rRNA fluorescence in situ hybridization (FISH), and for imaging of samples using confocal laser scanning microscopy (CLSM). Direct imaging is the only method which can definitively identify biofilms on implants and complements both culture and culture-independent diagnostic methods.

In vitro study of elution kinetics and bio-activity of meropenem-loaded acrylic bone cement.

BACKGROUND: Use of antibiotic-loaded acrylic bone cement to treat orthopaedic infections continues to remain popular, but resistance to routinely used antibiotics has led to the search for alternative, more effective antibiotics. We studied, in vitro, the elution kinetics and bio-activity of different concentrations of meropenem-loaded acrylic bone cement. METHODS: Meropenem-loaded bone cement cylinders of different concentrations were serially immersed in normal saline. Elution kinetics was studied by measuring the drug concentration in the eluate, collected at pre-determined intervals, by high-performance liquid chromatography. Bio-activity of the eluate of two different antibiotic concentrations was tested for a period of 3 weeks against each of the following organisms: Staphylococcus aureus ATCC 2593 (MSSA), Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, S. aureus ATCC 43300 (MRSA) and Klebsiella pneumoniae ATCC 700603 (ESBL). RESULTS: Meropenem elutes from acrylic bone cement for a period of 3-27 days depending on the concentration of antibiotic. Higher doses of antibiotic concentration resulted in greater elution of the antibiotic. The eluate was found to be biologically active against S. aureus ATCC 2593 (MSSA), P. aeruginosa ATCC 27853, E. coli ATCC 25922 and K. pneumoniae ATCC 700603 (ESBL) for a period of 3 weeks. CONCLUSIONS: The elution of meropenem is in keeping with typical antibiotic-loaded acrylic bone cement elution characteristics. The use of high-dose meropenem-loaded acrylic bone cement seems to be an attractive option for treatment of resistant Gram-negative orthopaedic infections but needs to be tested in vivo.

Supports used for positioning of patients in hip arthroplasty: is there an infection risk?

INTRODUCTION: Infection after joint arthroplasty is a disastrous complication. Implants used in hip arthroplasty increase the risk of infection from organisms of low pathogenicity. Potential reservoirs, that have not been assessed as yet, are the supports used for patient positioning in hip arthroplasty. The purpose of this study was to assess these supports for presence of bacterial pathogens. SUBJECTS AND METHODS: We studied 40 supports used in 20 hip arthroplasty procedures. Tryptone soya agar plates were used to sample these supports. All agar plates were incubated at 37 °C for 48 h. RESULTS: Of the 20 anterior supports, 17 (85%) showed bacterial colonisation; of the 20 posterior supports, 10 (50%) had bacterial colonisation. Fourteen (52%) supports were contaminated with one organism, 9 (33%) with two organisms, three (11%) with three organisms and one (4%) with four organisms. Coagulase-negative staphylococci were the most common isolated organisms (61%) followed by coryneforms (10%) and bacilli (10%). Anterior supports had two times more colony forming units compared to the posterior supports. CONCLUSIONS: This study showed contamination of supports used for positioning patients during hip arthroplasty. It reflects poor cleaning practice and certainly raises the possibility that a high bacterial load on these supports may contribute to higher infection rates in hip arthroplasties. The study raises concerns related to contamination of supports, as there is a potential for cross-infection, wound problems, and deep sepsis around implants which could be disastrous. While colonisation does not equate with infection, we suggest thorough cleaning of the supports before and after every surgical procedure.

Analysis of the microbial load in instruments used in orthopedic surgeries.

BACKGROUND: Because of advances in technology, the number of orthopedic surgeries, mainly hip and knee replacement surgeries, has increased, with a total of 150,000 prosthetic surgeries estimated per year in the United States and 400,000 worldwide. METHODS: We used an exploratory cross-sectional study, with a quantitative approach to determine the microbial load in instruments used in orthopedic surgeries, quantifying and identifying the microbial growth genus and species, according to the surgical potential of contamination that characterizes the challenge faced by the Material and Sterilization Center at the Institute of Orthopedics and Traumatology of Hospital das Clinicas of the School of Medicine of the University of Sao Paulo, Brazil.The orthopedic surgical instruments were immersed, after their use, in sterilized distilled water, sonicated in an ultrasonic washer, and posteriorly agitated. Subsequently, the wash was filtrated through a 0.45-mum membrane and incubated in aerobic and anaerobic mediums and in medium for fungi and yeasts. RESULTS: In clean surgeries, 47% of the instruments were contaminated; in contaminated surgeries, 70%; and, in infected surgeries, 80%. Regardless of the contamination potential of the surgeries, the highest quantitative incidence of microorganism recovery was located in the 1 to 100 colony-forming unit range, and 13 samples presented a microbial growth potential >300 colony-forming units. Regardless of the contamination potential of the surgeries, there was a convergence in the incidence of negative-coagulase Staphylococcus growth (28%, clean surgeries; 32%, contaminated surgeries; and 29%, infected surgeries) and Staphylococcus aureus (28%, contaminated surgeries; and 43%, infected surgeries). CONCLUSION: Most of the microorganisms recovered from the analyzed instruments (78%) were vegetative bacteria that presented their death curve at around 80 degrees C, characterizing a low challenge considering the processes of cleaning and sterilization currently employed by the Material and Sterilization Center. Fewer microorganisms were recovered from instruments used in clean surgeries in comparison with those used in contaminated and infected surgeries.

Potential for contamination of orthopaedic implants using individually wrapped screws.

BACKGROUND: There has been an anecdotal shift away from screw banks in orthopaedic surgery in preference for individually wrapped screws. Our hypothesis is that opening individually wrapped items introduces a possible source of contamination of the surgical set-up and repeated opening compounds this potential. Theoretically, this could pose a greater infection risk to the patient than the alternative of a screw bank. METHODS: One hundred screws were double wrapped in an identical fashion to the standard practice at our hospital. We used Glitterbug (Glitterbug cream is a commercially available product available from Brevis Corporation, Salt Lake City, Utah, USA) cream, which is designed to be near invisible to the naked eye but visible under ultraviolet light. After bathing their hands in the cream, 5 theatre nurses opened 20 screws as if being carried out during an operation. Ultraviolet light was used to evaluate transfer of the cream. Samples were considered positive if there was any evidence of fluorescence under ultraviolet light. Five screw banks were also opened and inspected using the same methods for comparison. RESULTS: We identified contamination in one of the 100 screws. No contamination of the inner packaging of the screw banks was observed. At no stage did any participants believe that contamination had occurred. CONCLUSIONS: This study has shown that the opening of individually wrapped items increases the risk for potential contamination of an operative field, suggesting that the move towards using individually wrapped components for surgery warrants further investigation. We believe that orthopaedic operating theatres should consider the use of screw banks to minimize the risk of contamination.

Gentamicin-loaded bioresorbable films for prevention of bacterial infections associated with orthopedic implants.

Adhesion of bacteria to biomaterials and the ability of many microorganisms to form biofilms on foreign bodies are well-established as major contributors to the pathogenesis of implant-associated infections. Treatment of bone infection remains problematic, due to the difficulty of systemically administered antibiotics to locally penetrate bone. The current research addresses this issue by focusing on the development and study of novel gentamicin-loaded bioresorbable films designed to serve as "coatings" for fracture fixation devices and prevent implant-associated infections. Poly(L-lactic acid) and poly (D,L-lactic-co-glycolic acid) films containing gentamicin were developed through solution processing. The effects of polymer type, drug content, and processing conditions on the drug release profile were studied with respect to film morphology. The examined films generally exhibited a burst effect followed by a moderate approximately constant rate of release. The drug contents in the surrounding medium exceeded the required minimal effective concentration. Various gentamicin concentrations that were released from the films with time exhibited efficacy against bacterial species known to be involved in orthopedic infections. The developed systems can be applied on the surface of any metallic or polymeric fracture fixation device, and may therefore comprise a significant contribution to the field of orthopedic implants.

Virulence factors in enterococcal infections of orthopedic devices.

Enterococci are opportunistic pathogens which today represent one of the leading causes of nosocomial infections. We have examined a collection of 52 Enterococcus faecalis isolated from orthopedic infections to determine if they were characterized by a specific pattern of virulence factors. The isolates were evaluated for biofilm formation, presence of genes coding the enterococcal surface protein (esp) and gelatinase (gelE), as well as for gelatinase production. While the rate of esp-positive isolates was comparable to that found among strains from other clinical sources, we found a significantly higher rate of strong biofilm formers and gelatinase producers. Particularly high was the rate of gelE-carrying strains expressing the gene. Data suggest that these two factors in particular may play an important role in enterococcal infections associated with biomaterials.

Automated ribotyping to distinguish the different non Sau/ non Sep staphylococcal emerging pathogens in orthopedic implant infections.

Several species belonging to Staphylococcus genus, other than Staphylococcus aureus and Staphylococcus epidermidis (non Sau/ non Sep species), exhibit increasing abilities as opportunistic pathogens in the colonisation of periprosthetic tissues. Consequently, the availability of means for accurate identification is crucial to assess the pathogenic characteristics and to clarify clinical relevance of the individual species. Here, 146 clinical staphylococcal isolates belonging to non Sau/ non Sep species from prosthesis-associated orthopedic infections were analyzed by conventional enzymatic galleries and by automated ribotyping. Twelve different species were recognised: S. capitis, S. caprae, S. cohnii, S. equorum, S. haemolyticus, S. hominis, S. lugdunensis, S. pasteuri, S. sciuri, S. simulans, S. warneri, S. xylosus. Ribotype identifications were compared with the phenotypes obtained by the Api 20 Staph system and/or ID 32 Staph system. ID 32 Staph profiles were more consistent with ribotyping results than Api Staph profiles. Across the different staphylococcal species investigated, correct identifications with Api Staph were 45%, while with ID 32 Staph they were 59%. It has, however, to be mentioned that ID 32 Staph was mostly applied to discriminate unmatched ribotyping and Api Staph identifications, thus to a subpopulation of strains with ""atypical"" metabolic profile. Automated ribotyping provided a correct identification for 91% of the isolates. These results confirm automated ribotyping as a convenient rapid technique, still subject to improvements, which will accurately and rapidly recognise the newly emerging staphylococcal pathogens in implant-related orthopedic infections.