Efficacy of Antibacterial Bioactive Glass S53P4 Against S. aureus Biofilms Grown on Titanium Discs In Vitro

ABSTRACT: 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 (0.5–0.8mm and <45 mm) were placed in contact with the discs containing biofilms. Glass beads (0.5mm) 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 <45mm was significantly higher than by BAG-S53P4 0.5–0.8mm. BAG-S53P4 has a clear growth-inhibitory effect on S. aureus biofilms. BAG-S53P4 <45mm is more efficient against biofilm growth in vitro comparing with BAG-S53P4 0.5–0.8mm. 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. 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:175–177, 2014

Effect of two cleaning processes for bone allografts on gentamicin impregnation and in vitro antibiotic release.

Bone allografts are a useful and sometimes indispensable tool for the surgeon to repair bone defects. Microbial contamination is a major reason for discarding allografts from bone banks. To improve the number of safe allografts, we suggest chemical cleaning of the grafts followed by antibiotic impregnation. Comparison of two chemical cleaning processes for bone allografts aiming for antibiotic impregnation and consequently delivery rates in vitro. Bone chips of 5–10 mm were prepared from human femoral heads. Two cleaning methods (cleaning A and cleaning B) based on solutions containing hydrogen peroxide, paracetic acid, ethanol and biological detergent were carried out and compared. After the cleaning processes, the bone chips were impregnated with gentamicin. Bacillus subtilis bioassay was used to determine the gentamicin release after intervals of 1–7 days. Differences were compared with non-parametric Mann–Whitney U tests. The zones of inhibition obtained from the bone grafts cleaned with both cleaning processes were similar between the groups. The concentration of the released antibiotic was decreasing gradually over time, following a similar pattern for both groups. The cleaning procedure A as well as the cleaning procedure B for bone allografts allowed the impregnation with gentamicin powder in the same concentrations in both groups. The delivery of gentamicin was similar for both groups. Both cleaning procedures were easy to be carried out, making them suitable for routine use at the bone banks

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. Methods and Results: Staphylococcus aureus was grown on polystyrene and on metal discs made of titanium and chrome–cobalt. Antibiotic susceptibility was assessed by examining the recovery of cells after antibiotic exposure and by measuring the biofilm inhibitory concentration (BIC). The minimal inhibitory concentration (MIC) was assessed with planktonic cells. Bacterial growth was examined by scanning electron microscopy. The antibiotic concentration for biofilm inhibition (BIC) was higher than the MIC for all antibiotics. Microscopic images showed the biofilm structure characterized by groups of cells covered by a film. Conclusions:  All models allowed biofilm formation and testing with several antibiotics in vitro. Gentamicin and rifampicin are the most effective inhibitors of Staph. aureus biofilm-related infections. We recommend MBEC™-HTP assay for rapid testing of multiple substances and TMZF® and CrCo discs for low-throughput testing of antibiotic susceptibility and for microscopic analysis. Significance and Impact of the Study: In vitro assays can improve the understanding of biofilms and help developing methods to eliminate biofilms from implant surfaces. One advantage of the TMZF® and CrCo discs as biofilm in vitro assay is that these metals are commonly used for orthopaedic implants. These models are usable for future periprosthetic joint infection studies

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

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

Influence of poly-N-acetylglucosamine in the extracellular matrix on N-chlorotaurine mediated killing of Staphylococcus epidermidis

N–chlorotaurine (NCT) has recently been shown to have bactericidal activity against bacterial biofilm on metal discs (Coraca-Huber et al., 2014). In a biofilm, Staphylococcus epidermidis polymerizes poly-N-acetylglucosamine (PNAG) to form an extracellular matrix (ECM). Pseudomonas aeruginosa does not express this PNAG and has been shown to be highly susceptible to NCT. We compared the action of NCT on S. epidermidis 1457, a PNAG positive strain (SE1457) and S. epidermidis 1457- M10 an isogenic PNAG negative mutant (SE1457 M10). NCT-mediated killing was more effective and quicker on the PNAG negative strain SE1457 M10. Bacteria hidden in biofilms for prolonged periods of time were generally more susceptible than freshly formed biofilms. The differences in NCT-mediated killing might not be direct effects since NCT did not react with the monomeric N-acetylglucosamine, but might be explained by denser growth in the PNAG-containing biofilm produced by the wild type strain, which results in delayed penetration of NCT. The higher susceptibility of older biofilms to NCTmediated killing could be explained by more pronounced 3D architecture and subsequent larger surface area for interactions with NCT

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

Disseminated Infection with Prototheca zopfii after Unrelated Stem Cell Transplantation for Leukemia

Disseminated infection with Prototheca zopfii is a rare disease in immunosuppressed patients. We here report the first case of lethal infection with P. zopfii following unrelated stem cell transplantation for leukemia. Breakthrough protothecosis occurred during long-term administration of voriconazole in the case of pulmonary aspergillosis