Débora C. Coraça-Huber

Efficacy of antibacterial bioactive glass S53P4 against S. aureus biofilms grown on titanium discs in vitro

We evaluated the effectiveness of different sizes of bioactive glass S53P4 against Staphylococcus aureus biofilms grown on metal discs in vitro. S. aureus biofilms were cultivated on titanium discs. BAG‐S53P4 (0.5–0.8 mm and <45 µm) were placed in contact with the discs containing biofilms. Glass beads (0.5 mm) were used as a control. After each interval, the pH from each sample was measured. Colony forming units were counted for the biofilm recovery verification. In parallel, we tested the activity of bioactive glass against S. aureus planktonic cells. We found that BAG‐S53P4 can suppress S. aureus biofilm formation on titanium discs in vitro. The suppression rate of biofilm cells by BAG‐S53P4 <45 µm was significantly higher than by BAG‐S53P4 0.5–0.8 mm. BAG‐S53P4 has a clear growth‐inhibitory effect on S. aureus biofilms. BAG‐S53P4 <45 µm is more efficient against biofilm growth in vitro comparing with BAG‐S53P4 0.5–0.8 mm. Bioactive glass S53P4 has potential to be used as bone substitute for the resolution of infection complications in joint replacement surgeries and treatment of chronic osteomyelitis.

Evaluation of MBEC™‐HTP biofilm model for studies of implant associated infections

The bacteria in implant‐related infections can evade host defenses by forming biofilms. The more we understand biofilm behavior, the better we can fight against then clinically. In vitro models for biofilms allow tests simulating in vivo conditions. In this study we evaluated the Minimum Biofilm Eradication Concentration‐High Throughput Plates (MBEC™‐HTP) as biofilm in vitro model for studies of implant associated infections. Staphylococcus aureus and Staphylococcus epidermidis biofilms were grown on MBEC™‐HTP. To ensure the biofilm formation, antibiotic susceptibility tests and scanning electron microscopy (SEM) was carried out. Susceptibility tests were carried out using gentamicin, vancomycin, rifampicin, fosfomycin, clindamycin, and linezolid. Colony forming units counting were carried out. Minimal inhibitory concentration (MIC) and biofilm inhibitory concentration (BIC) were estimated. The CFU counting showed potency of rifampicin and daptomycin against S. epidermidis biofilms and rifampicin against S. aureus biofilms. SEM images showed proteic material in contact with cells. The differences between BIC and MIC demonstrated the biofilm formation as well as the SEM images. Rifampicin and daptomycin are good choices against biofilm related infections. Moreover, after suggested modifications, the model used in this study is eligible to further studies of implant associated infections.

Staphylococcus aureus biofilm formation and antibiotic susceptibility tests on polystyrene and metal surfaces

Aim:  We compared the MBEC™‐HTP assay plates made of polystyrene with metal discs composed of TMZF® and CrCo as substrates for biofilm formation.

Effect of freezing on the release rate of gentamicin palmitate and gentamicin sulfate from bone tissue

In this study we evaluated gentamicin palmitate salt and gentamicin sulfate salt mixed with bone chips after storage at −80°C. Different concentration rates of gentamicin sulfate and gentamicin palmitate were mixed with human bone chips and stored for 1–6 months at −80°C. Nonstored samples were used as control. The release of the antibiotics from the bone was carried out in phosphate‐buffered saline. Antibiotic concentrations in the elutions were determined with microbiological agar diffusion assay using Bacillus subtilis. Susceptibility tests were carried out using Staphylococci strains. The rate of gentamicin base (GB) released from bone was similar for all gentamicin salts and all storage conditions. The elutions released were efficient on reducing S. aureus and S. epidermidis CFU during all storage time. In resume, the capacity of bone grafts to act as gentamicin carriers has been confirmed in this study. GS + GP showed equivalent efficacy against S. aureus and S. epidermidis compared with GS pure. The lower delivery rate of GS + GP, related to its affinity with fat tissue can be an advantage for longer release times, increasing the local protection against infections. Storage at −80°C does not interfere on the gentamicin salts activity used.

Calcium Carbonate Powder Containing Gentamicin for Mixing With Bone Grafts

Bone grafts are used for reconstructing bone defects caused by implant-associated complications, trauma, and tumors. Surgery with bone allografts is complex and time consuming; therefore, it is prone to a higher infection rate (2.0%-2.5%). In the case of site infection, systemically administered antibiotics cannot reach the infected bone graft. This study evaluated the use of resorbable bone graft substitute powder (HERAFILL; Heraeus Medical GmbH, Wehrheim, Germany) as a bone void-filling material as well as an antibiotic carrier for mixing with bone grafts. The antibiotic activity of the bone chips mixed with HERAFILL powder was measured by drug release tests and bacterial susceptibility with Bacillus subtilis, Staphylococcus epidermidis, and Staphylococcus aureus. HERAFILL powder was added to the bone chips (bone chips/HERAFILL; w/w = 1:1), mixed with a spatula, and vortexed for 1 minute. Gentamicin base release was evaluated in phosphate-buffered saline for up to 7 days using B subtilis bioassay. Antimicrobial efficacy was tested with S aureus and S epidermidis. The average amount of gentamicin base released from bone chips mixed with HERAFILL at 0 to 12 hours was 99.66 mg/mL. On day 7, the gentamicin base released 0.42 mg/mL. The elution released from bone chips mixed with HERAFILL promoted the formation of a zone of inhibition on S epidermidis and S aureus plates. This study confirmed the capacity of bone grafts to act as antibiotic carriers once mixed with HERAFILL powder. Bone chips mixed with HERAFILL showed efficacy against S aureus and S epidermidis.

Optimizing the grain size distribution of allografts in bone impaction grafting

In bone impaction grafting, allografts in the form of bone chips are used for reconstruction of defects and to induce bone remodeling. Optimizing grain size distribution of this allograft material should help prevent implant subsidence by achieving higher primary stability of the graft. We evaluated the influence of grain size distribution on the mechanical stability of allograft material. Bone tissue was rinsed, and the grain size distribution of the allograft material was determined by performing a sieve analysis. Uniaxial compression tests were carried out before and after a standardized compaction procedure for samples with controlled grain size distribution and a control group. Allografts with controlled grain size distribution showed a yield limit almost twice as high as in the control group after a standardized compaction procedure. A better interlocking between bone particles was observed compared to the control group. Thus, grain size distribution has a major impact on the mechanical stability of bone grafts. By controlling the grain size distribution of allograft material, a tighter packing can be achieved and subsequently implant subsidence of implants could be avoided.

Bactericidal Activity of N-Chlorotaurine against Biofilm-Forming Bacteria Grown on Metal Disks

ABSTRACT Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies.

Lyophilized allogeneic bone tissue as an antibiotic carrier

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.

Pseudomonas aeruginosa outcompetes other bacteria in the manifestation and maintenance of a biofilm in polyvinylchloride tubing as used in dental devices.

Abstract In a PVC tube as a model system for dental devices, Pseudomonas aeruginosa outcompetes Staphylococcus aureus and Klebsiella pneumoniae for the biofilm formation. P. aeruginosa has advantage over the other strains due to higher tolerance for low-nutrient situations or direct killing by the production of soluble factors like pyocyanin.

Effect of storage temperature and antibiotic impregnation on the quantity of bone morphogenetic protein seven in human bone grafts

PurposeThe aim of this study was to quantify the amount of bone morphogenic protein 7 (BMP-7) in bone samples in different storage and treatment conditions used in bone banks and thereby evaluate the benefit of this test as a routine measure before bone grafting.MethodsFresh as well as frozen bone chips, each with and without antibiotic impregnation, were screened for their BMP-7 content. Human bone chips were produced from femoral heads of two female donors who had undergone total hip replacement surgery. The amount of BMP-7 was detected using a commercially available enzyme-linked immunosorbent assay (ELISA) test.ResultsThere were no significant differences between groups in samples obtained from the first femoral head. Bone-chip samples derived from the second femoral head showed significant differences between groups. The actual amount of these differences was small and most likely biologically irrelevant. It is important to note that there was a significant difference between groups when comparing both femoral heads, reflecting donor-to-donor variability.ConclusionELISA testing for BMP-7 as a qualitative measurement of bone grafts should be considered a routine quality-control test for bone banks.