Prevention of Propionibacterium acnes biofilm formation in prosthetic infections in vitro.

BACKGROUND: The role of Propionibacterium acnes in shoulder arthroplasty and broadly in orthopedic prosthetic infections has historically been underestimated, with biofilm formation identified as a key virulence factor attributed to invasive isolates. With an often indolent clinical course, P acnes infection can be difficult to detect and treat. This study investigates absorbable cements loaded with a broad-spectrum antibiotic combination as an effective preventive strategy to combat P acnes biofilms. METHODS: P acnes biofilm formation on an unloaded synthetic calcium sulfate (CaSO4) bone void filler cement bead was evaluated by scanning electron microscopy over a period of 14 days. Beads loaded with tobramycin alone or vancomycin alone (as comparative controls) and beads loaded with a vancomycin-tobramycin dual treatment were assessed for their ability to eradicate planktonic P acnes, prevent biofilm formation, and eradicate preformed biofilms using a combination of viable-cell counts, confocal microscopy, and scanning electron microscopy. RESULTS: P acnes surface colonization and biofilm formation on unloaded CaSO4 beads was slow. Beads loaded with antibiotics were able to kill planktonic cultures of 106 colony-forming units/mL, prevent bacterial colonization, and significantly reduce biofilm formation over periods of weeks. Complete eradication of established biofilms was achieved with a contact time of 1 week. CONCLUSIONS: This study demonstrates that antibiotic-loaded CaSO4 beads may represent an effective antibacterial and antibiofilm strategy to combat prosthetic infections in which P acnes is involved.

Gene Expression Measurement Module (GEMM) for space application: Design and validation.

In order to facilitate studies on the impact of the space environment on biological systems, we have developed a prototype of GEMM (Gene Expression Measurement Module) - an automated, miniaturized, integrated fluidic system for in-situ measurements of gene expression in microbial samples. The GEMM instrument is capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing the RNA to probes attached to a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. To function on small, uncrewed spacecraft, the conventional, laboratory protocols for both sample preparation and hybridization required significant modifications. Biological validation of the instrument was carried out on Synechococcus elongatus, a photosynthetic cyanobacterium known for its metabolic diversity and resilience to adverse conditions. It was demonstrated that GEMM yielded reliable, reproducible gene expression profiles. GEMM is the only high throughput instrument that can be deployed in near future on space platforms other than the ISS to advance biological research in space. It can also prove useful for numerous terrestrial applications in the field.

In Vitro Efficacy of Antibiotics Released from Calcium Sulfate Bone Void Filler Beads.

15 different antibiotics were individually mixed with commercially available calcium sulfate bone void filler beads. The antibiotics were: amikacin, ceftriaxone, cefuroxime, ciprofloxacin, clindamycin, colistamethate sodium, daptomycin, gentamicin, imipenem/cilastatin, meropenem, nafcillin, rifampicin, teicoplanin, tobramycin and vancomycin. The efficacy of specific released antibiotics was validated by zone of inhibition (ZOI) testing using a modified Kirby⁻Bauer disk diffusion method against common periprosthetic joint infection pathogens. With a subset of experiments (daptomycin, rifampin, vancomycin alone and rifampin and vancomycin in combination), we investigated how release varied over 15 days using a repeated ZOI assay. We also tested the ability of these beads to kill biofilms formed by Staphylococcus epidermidis 35984, a prolific biofilm former. The results suggested that certain antibiotics could be combined and released from calcium sulfate with retained antibacterial efficacy. The daptomycin and rifampin plus vancomycin beads showed antimicrobial efficacy for the full 15 days of testing and vancomycin in combination with rifampin prevented resistant mutants. In the biofilm killing assay, all of the antibiotic combinations showed a significant reduction in biofilm bacteria after 24 h. The exposure time was an important factor in the amount of killing, and varied among the antibiotics.

Application of Calcium Sulfate for Dead Space Management in Soft Tissue: Characterisation of a Novel In Vivo Response.

Management of dead space (DS) is a fundamental aspect of surgery. Residual DS following surgery can fill with hematoma and provide an environment for bacterial growth, increasing the incidence of postoperative infection. Materials for managing DS include polymethyl-methacrylate (PMMA), which is nonresorbing and requires removal in a second surgical procedure. The use of calcium sulfate (CS) offers the advantage of being fully absorbed and does not require subsequent surgical removal. As CS has historically been used as a bone void filler, there are some concerns for the risk of heterotopic ossification (HO) when implanted adjacent to soft tissue. This study assessed the osteoinductive potential of CS and identified and characterised residual material present in muscle tissue using histology, energy-dispersive X-ray spectroscopy analysis, and scanning electron microscopy (SEM). CS beads with and without antibiotic were implanted in intramuscular sites in both athymic rats and New Zealand white rabbits. At 28 days after implantation in the rat model, no signs of osteoinduction were observed. In the rabbit model, at 21 days after implantation, almost complete bead absorption and presence of a "halo" of material in the surrounding muscle tissue were confirmed. Our results suggested that the halo of material was a calcium phosphate precipitate, not HO.

Influence of third-body particles originating from bone void fillers on the wear of ultra-high-molecular-weight polyethylene.

Calcium sulfate bone void fillers are increasingly being used for dead space management in infected arthroplasty revision surgery. The presence of these materials as loose beads close to the bearing surfaces of joint replacements gives the potential for them to enter the joint becoming trapped between the articulating surfaces; the resulting damage to cobalt chrome counterfaces and the subsequent wear of ultra-high-molecular-weight polyethylene is unknown. In this study, third-body damage to cobalt chrome counterfaces was simulated using particles of the calcium sulfate bone void fillers Stimulan(®) (Biocomposites Ltd., Keele, UK) and Osteoset(®) (Wright Medical Technology, TN, USA) using a bespoke rig. Scratches on the cobalt chrome plates were quantified in terms of their density and mean lip height, and the damage caused by the bone void fillers was compared to that caused by particles of SmartSet GMV PMMA bone cement (DePuy Synthes, IN, USA). The surface damage from Stimulan(®) was below the resolution of the analysis technique used; SmartSet GMV caused 0.19 scratches/mm with a mean lip height of 0.03 µm; Osteoset(®) led to a significantly higher number (1.62 scratches/mm) of scratches with a higher mean lip height (0.04 µm). Wear tests of ultra-high-molecular-weight polyethylene were carried out in a six-station multi-axial pin on plate reciprocating rig against the damaged plates and compared to negative (highly polished) and positive control plates damaged with a diamond stylus (2 µm lip height). The wear of ultra-high-molecular-weight polyethylene was shown to be similar against the negative control plates and those damaged with third-body particles; there was a significantly higher (p < 0.001) rate of ultra-high-molecular-weight polyethylene wear against the positive control plates. This study showed that bone void fillers of similar composition can cause varying damage to cobalt chrome counterfaces. However, the lip heights of the scratches were not of sufficient magnitude to increase the wear of ultra-high-molecular-weight polyethylene above that of the negative controls.

Biofilm prevention of gram-negative bacterial pathogens involved in periprosthetic infection by antibiotic-loaded calcium sulfate beads in vitro.

Biofilm formation represents a key stage in the pathogenesis of prosthetic infections (PIs). More tolerant to antibiotics than their planktonic counterparts, biofilm bacteria are difficult to eradicate using conventional therapeutic regimes. A common approach in PI management is the adjunctive use of localised antibiotics in addition to systemic administration in an attempt to protect the implant from colonisation by infiltrating bacteria. This study evaluates the antibacterial and antibiofilm efficacy of antibiotic-loaded dissolvable calcium sulphate, previously shown to be effective against key gram-positive pathogens, against gram-negative species important in the establishment of chronic infection in PIs. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin and both antibiotics in combination were assessed for their ability to eradicate planktonic Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae strains. The efficacy of the beads in preventing biofilm formation and eliminating established biofilms over multiple days was evaluated using confocal laser scanning microscopy (CSLM) imaging combined with image analysis and viable cell counts. Beads loaded with antibiotics demonstrated effective eluting concentrations for up to 37 d depending on the bacterial strain. In the presence of repeated bacterial challenges, antibiotic-loaded beads prevented bacterial colonisation and significantly reduce biofilm formation for the duration of the assay (7 d). Complete eradication of established biofilms was more difficult with evidence of biofilm regrowth after 1 week of contact with antibiotic-loaded beads, despite data suggesting a complete kill was achieved at earlier timepoints of 24 h and 72 h in the case of K. pneumoniae and P. aeruginosa. This study provides further evidence that calcium sulfate beads loaded with vancomycin and tobramycin may be a useful adjunctive component to the successful management of PIs.

Local release of antibiotics for surgical site infection management using high-purity calcium sulfate: an in vitro elution study.

BACKGROUND: The aim of this study was to characterize the elution of four antibiotics from pharmaceutical-grade calcium sulfate beads and show that the eluted antibiotics retained efficacy. METHODS: Calcium sulfate was combined with gentamicin, tobramycin, vancomycin, or rifampicin (ratio: 20 g of calcium sulfate, to 240 mg, 500 mg, 900 mg, and 600 mg of antibiotic, respectively). Three grams of beads were immersed in 4 mL of sterile phosphate-buffered saline (PBS) at 37°C. At each time point (4, 8, 24 h; 2, 7, 14, 28, 42 d), eluates were removed for analysis by liquid chromatography-mass spectrometry. The antimicrobial efficacy of antibiotics combined with calcium sulfate beads after 42 d was tested by a modified Kirby-Bauer disc diffusion assay. RESULTS: All samples showed a generally exponential decay in the eluted antibiotic concentration. At the first time point, both gentamicin and tobramycin had eluted to a peak concentration of approximately 10,000 mcg/mL. For rifampicin, the peak concentration occurred at 24 h, whereas for vancomycin, it occurred at 48 h. The eluted concentrations exceeded the minimum inhibitory concentration for common periprosthetic joint infection pathogens for the entire span of the 42 study days. Mass spectrometry confirmed all antibiotics were unchanged when eluted from the calcium sulfate carrier. Antimicrobial efficacy was unaltered after 42 d in combination with calcium sulfate at 37°C. CONCLUSIONS: Pharmaceutical-grade calcium sulfate has the potential for targeted local release of tobramycin, gentamicin, vancomycin, and rifampicin over a clinically meaningful time period.

Development of a Novel Model for the Assessment of Dead-Space Management in Soft Tissue.

Following extensive surgical debridement in the treatment of infection, a "dead space" can result following surgical closure that can fill with hematoma, an environment conducive to bacterial growth. The eradication of dead space is essential in order to prevent recurrent infection. This study describes a novel small animal model to investigate dead-space management in muscle tissue. Two absorbable test materials were implanted in each animal; beads of calcium sulfate alone, and beads loaded with vancomycin and tobramycin. In-life blood samples and radiographs were taken from each animal following implantation. Animals were sacrificed at 1, 7, 21, 42, and 63 days post-operatively (n = 4), and implant sites were analysed by micro-computed tomography, histology and immunohistochemistry. Complete resorption was confirmed radiographically at 3 weeks post-implantation. Histologically, the host tissue response to both materials was identical, and subsequent healing at the implant sites was observed with no dead space remaining. Vancomycin was not detected in blood serum. However, peak tobramycin levels were detected in all animals at 6 hours post-implantation with no detectable levels in any animals at 72 hours post implantation. Serological inflammatory cytokine expression for IL-6, TNF-α and IL-1ß indicated no unusual inflammatory response to the implanted materials or surgical procedure. The model was found to be convenient and effective for the assessment of implant materials for management of dead space in muscle tissue. The two materials tested were effective in resolving the surgically created dead space, and did not elicit any unexpected adverse host response.

PK11195, a peripheral benzodiazepine receptor ligand, chemosensitizes acute myeloid leukemia cells to relevant therapeutic agents by more than one mechanism.

Like Bcl-2, peripheral benzodiazepine receptors (pBzRs) reside in mitochondrial pores, are frequently over-expressed in tumor cells, and can protect cells from apoptotic cell death. We now show that the high-affinity, pBzR-specific ligand, PK11195, chemosensitizes AML cells to relevant chemotherapeutics, but is relatively non-toxic as a single agent, and does not chemosensitize normal myeloid cells. PK11195 can block p-glycoprotein efflux in AMLs, contributing to increased daunomycin toxicity in efflux-competent AMLs, but can also sensitize AMLs to cytarabine and DNR-sensitize efflux-incompetent AMLs, presumably via mitochondrial pore effects documented in other models. Therefore, PK11195 might contribute to improved clinical outcomes in AML.

Body mass index-associated differences in response to ovulation induction with letrozole.

OBJECTIVE: To compare occurrence of pregnancy among obese (body mass index [BMI] ≥30) and nonobese (BMI <30), infertile women undergoing ovulation induction with the aromatase inhibitor letrozole followed by intrauterine insemination (IUI). DESIGN: Retrospective cohort study. SETTING: Academic reproductive endocrinology and infertility clinic. PATIENT(S): Ninety women with a variety of infertility diagnoses. INTERVENTION(S): Letrozole (5 mg) on menstrual cycle days 3-7, followed by intrauterine insemination (IUI). MAIN OUTCOME MEASURE(S): Occurrence of pregnancy and pregnancy outcomes. RESULT(S): Ninety women underwent 180 letrozole-IUI cycles. Conception of pregnancy occurred in 10.4% and 18.2% of the BMI <30 and BMI ≥30 groups, respectively. Using BMI as a continuous variable showed a pregnancy odds ratio of 1.093 (confidence interval 1.008-1.184) for each unit increase in BMI. Incidence of miscarriage, multiple births, number of mature follicles, and presence of LH surge were similar between groups. CONCLUSION(S): Our study of 90 women undergoing letrozole-IUI treatment showed greater likelihood of pregnancy in higher-BMI women, although the difference was not significant. Letrozole is an effective ovulation induction agent in higher-BMI women.