ROS induced bactericidal activity of amorphous Zn-doped titanium oxide coatings and enhanced osseointegration in bacteria-infected rat tibias.

Titanium-based endosseous implants with high antibacterial and osseointegration activities are extremely required in clinics. To achieve this line, herein the doped coatings with three kinds of Zn doses were micro-arc oxidized (MAOed) on Ti. They were examined to reveal a bilayered structure, in which the outer layer consisted completely of the amorphism comprising elements of Ti, O and Zn with Zn doped in the form of weaken Zn-O bonds, and the underlying layer was partially crystallized with nanocrystalline TiO2 and Zn2TiO4 to embed an amorphous matrix. While the Zn doped doses of the surface amorphous layers increased with elevating the MAOed voltages, the weaken Zn-O bonds in the amorphism were identified to act as both the contributor of Zn2+ controllable release and the generator of reactive oxide species (ROS) on the coatings. The enhanced HO• and O2-• formation on the elevated voltage MAOed coatings caused serious break of the cell walls and plasma membranes of S. aureus. In parallel, the enhanced Zn2+ release and extracellular H2O2 formation led to the enhanced intracellular ROS level of S. aureus, further aggravating the damage of plasma membrane, resulting in bacteria death. On contrary to the overdose of Zn doped coating, the moderate doses of Zn doped coatings did not induce additional intracellular ROS and attenuate viability and proliferation of osteoblasts in vitro, and promoted osseointegration in both S. aureus-uninfected and infected rat tibias, which ascribed to the strong antibacterial activity and un-attenuated cell function of the coatings in the infected case. STATEMENT OF SIGNIFICANCE: (1) The Zn-doped coatings revealed a bilayered structure of the surface layer comprising the Ti, O and Zn constructed amorphism with Zn in the form of weaken Zn-O bonds, and the underlying layer comprising nanocrystalline TiO2 and Zn2TiO4 to embed amorphous matrix. (2) The weaken Zn-O bonds in the amorphism were identified to act as both the contributor of Zn2+ controllable release and the generator of ROS on the coatings. (3) The enhanced Zn2+ release and ROS formation on the coatings killed S. aureus by inducing serious break of their cell walls and plasma membranes. This effect in combination of un-attenuated osteoblast proliferation endowed the moderate Zn doped coatings with enhanced osseointegration in S. aureus-infected rat tibias.

Early outcome of culture-negative infection in open fractures of the lower limb: A prospective study.

Background: Culture-negative infections in open long bone fractures are frequently encountered in clinical practice. We aimed to identify the rate and outcome of culture-negative infections in open long bone fractures of lower limb. Methodology: A prospective cohort study was conducted from November 2015 to May 2017 on Gustilo and Anderson Grade III open long bone fractures of the lower limb. Demographic data, injury details, time from injury to receiving antibiotics and index surgical procedure were noted. Length of hospital stay, number of additional surgeries and occurrence of complications were also noted. Patients with infected open fractures were grouped as culture positive or culture negative depending on the isolation of infecting microorganisms in deep intraoperative specimen. The clinical outcome of these two groups was statistically analysed. Results: A total of 231 patients with 275 open fractures involving the femur, tibia or fibula were studied. There was clinical signs of infection in 84 patients (36.4%) with 99 fractures (36%). Forty-three patients (51.2%) had positive cultures and remaining 41 patients had negative cultures (48.8%). The rate of culture-negative infection in open type III long bone fractures in our study was 17.7%. There was no statistical difference in the clinical outcome between culture-negative and culture-positive infections. Conclusion: Failure to identify an infective microorganism in the presence of clinical signs of infection is routinely seen in open fractures and needs to be treated aggressively.

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.).

Ustilago echinata: Infection in a Mixed Martial Artist Following an Open Fracture.

Ustilago, a common fungal parasite of grains, is infrequently isolated as a pathogen in humans. We describe a case of Ustilago echinata infection following an open distal tibia fracture, review the current literature of this genus as a cause of invasive fungal infection in humans, and discuss management issues.

Comparison of Borate Bioactive Glass and Calcium Sulfate as Implants for the Local Delivery of Teicoplanin in the Treatment of Methicillin-Resistant Staphylococcus aureus-Induced Osteomyelitis in a Rabbit Model.

There is growing interest in biomaterials that can cure bone infection and also regenerate bone. In this study, two groups of implants composed of 10% (wt/wt) teicoplanin (TEC)-loaded borate bioactive glass (designated TBG) or calcium sulfate (TCS) were created and evaluated for their ability to release TEC in vitro and to cure methicillin-resistant Staphylococcus aureus (MRSA)-induced osteomyelitis in a rabbit model. When immersed in phosphate-buffered saline (PBS), both groups of implants provided a sustained release of TEC at a therapeutic level for up to 3 to 4 weeks while they were gradually degraded and converted to hydroxyapatite. The TBG implants showed a longer duration of TEC release and better retention of strength as a function of immersion time in PBS. Infected rabbit tibiae were treated by debridement, followed by implantation of TBG or TCS pellets or intravenous injection with TEC, or were left untreated. Evaluation at 6 weeks postimplantation showed that the animals implanted with TBG or TCS pellets had significantly lower radiological and histological scores, lower rates of MRSA-positive cultures, and lower bacterial loads than those preoperatively and those of animals treated intravenously. The level of bone regeneration was also higher in the defects treated with the TBG pellets. The results showed that local TEC delivery was more effective than intravenous administration for the treatment of MRSA-induced osteomyelitis. Borate glass has the advantages of better mechanical strength, more desirable kinetics of release of TEC, and a higher osteogenic capacity and thus could be an effective alternative to calcium sulfate for local delivery of TEC.

Efficacy of fosfomycin in experimental osteomyelitis due to methicillin-resistant Staphylococcus aureus.

The activity of fosfomycin was evaluated in an experimental methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis model. Eighteen rats were treated for 4 weeks with 150 mg of fosfomycin/kg of body weight intraperitoneally once daily or with saline placebo. After treatment, animals were euthanized and the infected tibiae were processed for quantitative bacterial culture. Bone cultures were positive for methicillin-resistant S. aureus in all 9 (100%) untreated controls and in 2 of 9 (22.2%) fosfomycin-treated rats. Thus, fosfomycin treatment was significantly more efficacious than placebo. No development of resistance was observed after the 4-week treatment period.

Gatifloxacin biodegradable implant for treatment of experimental osteomyelitis: in vitro and in vivo evaluation.

Osteomyelitis is an inflammatory bone disease caused by pyogenic bacteria. The advantages of localized biodegradable therapy for osteomyelitis include high local antibiotic concentration at the site of infection and obviation of the need for removal of the implant after treatment. The purpose of this study was to develop and evaluate a biodegradable implantable delivery system containing gatifloxacin (GAT) for the localized treatment of osteomyelitis, experimentally induced by methicillin resistant Staphylococcus aureus (MRSA). Implants, prepared by solvent casting technique, showed reasonable tensile strength. DSC examination indicated that GAT is present in an amorphous form in the implant. The in vitro release of GAT showed a profile characterized by an initial burst followed by a second stage of gradual delivery over 27 days. The in vivo release study revealed that GAT concentrations achieved during the first 3 weeks after implantation exceeded the MIC of GAT against MRSA by > 100,000 times. Bacterial tibial bone count performed in rabbits tibia 2 and 4 weeks after implantation of GAT implant in infected bone indicated complete eradication of infection in all treated rabbits as indicated by the significant decrease in bacterial count. The results show that the proposed implant may have a promising role in the therapeutic approach to osteomyelitis.

Interleukin-1, interleukin-6, and interleukin-10 responses after antibiotic treatment in experimental chronic Staphylococcus aureus osteomyelitis.

BACKGROUND: Staphylococcal implant infections' response to treatment may be correlated with cytokine production. We investigated the effect of certain antibiotics on the cytokine response in experimental osteomyelitis. METHODS: A stainless steel needle with an adherent slime-producing Staphylococcus aureus was implanted intramedullarly in the left tibia of 40 adult male Wistar rats. At 42 days after implantation, cefuroxime, vancomycin, tobramycin, and ciprofloxacin were administered intramuscularly every 12 h for 21 days. The control group was given no antibiotic. At the end of the treatment, implants and tibias were retrieved, and the bacterial numbers were estimated. Cytokines [interleukin-1alpha (IL-1alpha), IL-6, and IL-10] were determined (ELISA) in the tibial extract. RESULTS: Vancomycin and cefuroxime inhibited bone colonization in all tibias, and tobramycin and ciprofloxacin inhibited it only partially. Cefuroxime reduced the number of bacteria that adhered to the implants more than the other antibiotics. IL-1alpha and IL-6 showed higher levels in the ciprofloxacin-treated group than in the cefuroxime-treated and control groups. IL-6 levels in rats treated with cefuroxime were lower than in rats treated with tobramycin or vancomycin and the control group. Cefuroxime decreased IL-10 levels more than ciprofloxacin or vancomycin or those seen in the control group. CONCLUSIONS: The cefuroxime group showed the greatest decrease of pro-inflammatory cytokines. Different antibiotics produce different cytokine reactions that should be studied to choose the best treatment.

Efficacy of ciprofloxacin-releasing bioabsorbable osteoconductive bone defect filler for treatment of experimental osteomyelitis due to Staphylococcus aureus.

The concept of local antibiotic delivery via biodegradable bone defect fillers with multifunctional properties for the treatment of bone infections is highly appealing. Fillers can be used to obliterate surgical dead space and to provide targeted local bactericidal concentrations in tissue for extended periods. Eventually, the osteoconductive component of the filler could guide the healing of the bone defect. The present experimental study was carried out to test this concept in a localized Staphylococcus aureus osteomyelitis model in the rabbit (n = 31). A metaphyseal defect of the tibia was filled with a block of bone cement, followed by insertion of a bacterial inoculum. After removal of the bone cement and surgical debridement at 2 weeks, the defect was filled with a ciprofloxacin-containing (7.6% +/- 0.1%, by weight) composite (treated-infection group) or with a composite without antibiotic (sham-treated group). Both a positive control group (untreated-infection group) and a negative control group were also produced. The treatment response, monitored by positron emission tomography (PET) with fluorine-18-labeled fluorodeoxyglucose ([18F]FDG) at 3 and 6 weeks, showed rapidly decreasing amounts of [18F]FDG uptake in the treated-infection group (P = 0.001 compared with the results for the untreated-infection group at 6 weeks). The bacteriological analysis confirmed the eradication of the bone pathogen in the treated-infection group. However, three animals had culture-positive soft tissue infections. All animals in the sham-treated and untreated-infection groups had culture-positive bone infections with typical radiographic changes of osteomyelitis. Histomorphometry, peripheral quantitative computed tomography, and backscattered electron imaging of scanning electron microscopy images verified the osteoconductive properties of the bioactive glass microspheres within the composite. The median bone ciprofloxacin concentrations were 1.2 and 2.1 microg/g at two anatomic locations of the tibia. This is the first report to show the value of [18F]FDG PET for quantitative monitoring of the treatment response in bone infections. The collaborative results of bacteriologic and [18F-FDG] PET studies showed that use of the multifunctional composite was successful for eradication of the S. aureus pathogen from bone.

Treatment of infected tibial nonunion with tobramycin-impregnated calcium sulfate: report of two cases.

The treatment of infected tibial nonunion usually includes a staged reconstruction protocol. We present 2 patients with tibial nonunion and plate loosening with oxacillin-resistant Staphylococcus aureus infection. The patients were treated using the removal of the plate, radical debridement, and implantation of gentamycin-impregnated cement beads during the first stage. During the second stage, plate fixation was performed and tobramycin-impregnated calcium sulfate (Osteoset T) was used as a bone graft substitute. Neither an autogenous bone graft nor an allograft was used. At 3 years of follow-up, each tibia showed good union, and there was no recurrence of infection. We consider tobramycin-impregnated calcium sulfate to be an alternative method of bone grafting to treat infected tibial nonunion.

A simple infection model using pre-colonized implants to reproduce rat chronic Staphylococcus aureus osteomyelitis and study antibiotic treatment.

Staphylococcus aureus biofilms formed on medical implants represent a serious problem, being difficult to eradicate with antibiotic therapy and leading to chronic infections. Simplified in vivo and in vitro antibiotic susceptibility assays using biofilm bacteria are needed. In this work, a novel chronic osteomyelitis infection model was developed in rats in the absence of bacterial suspension, requiring the use of only 10(6) bacteria in biofilms at the site of surgery, with a full success in reproducing infection. Stainless-steel implants pre-colonized for 12 h with a highly adherent S. aureaus isolate were introduced into the rat tibiae. In animals not submitted to antibiotic treatment, infection was found in the implants and spread to bone in all cases, indicating the high efficacy of the model to reproduce osteomyelitis. The effect of a 21-day treatment with cefuroxime, vancomycin, tobramycin or ciprofloxacin on infection was studied in this model 42 days after surgery. Bone colonization was inhibited by vancomycin and cefuroxime. Cefuroxime (the most efficient antibiotic, able to sterilize 1 out of 8 implants) reduced the number of bacteria in biofilms adhered to implants at a higher extent than vancomycin, trobramycin and ciprofloxacin. Analogous observations were made in this work in vivo and in vitro on the relative antibiotic efficacy against S. aureus biofilm bacteria. suggesting the usefulness of both tests as a potential tool to study antibiotic suceptibility, and the need for new antimicrobials against these bacteria.

Therapy with hyperbaric oxygen for experimental osteomyelitis due to Staphylococcus aureus in rabbits.

Hyperbaric oxygen (HBO) is used as adjunctive therapy of chronic osteomyelitis, but its efficacy remains controversial. A recently developed rabbit model for osteomyelitis due to Staphylococcus aureus was used to compare the results of treatment with HBO, cephalothin, a combination of both, or no treatment. Cultures of bone were positive in 10 (91%) of 11 control animals (untreated), five (36%) of 14 animals treated with HBO, eight (47%) of 17 treated with cephalothin, and six (40%) of 15 treated with HBO plus cephalothin. All three treatment groups differed significantly from untreated controls in the number of positive cultures obtained (P less than 0.01), but there were no significant differences among treatment groups. In vitro growth and killing curves (1.0 microgram of cephalothin/ml) constructed after exposure to HBO revealed no change from parallel control studies in ambient air. These data demonstrate that therapy with HBO is at least as effective as antibiotic therapy. The therapeutic effectiveness of HBO does not appear to be related to antibacterial activity.