We previously reported the development of an osteogenic bone filler scaffold consisting of degradable polyurethane, hydroxyapatite, and decellularized bovine bone particles. The current study was aimed at evaluating the use of this scaffold as a means of local antibiotic delivery to prevent infection in a bone defect contaminated with Staphylococcus aureus. We evaluated two scaffold formulations with the same component ratios but differing overall porosity and surface area. Studies with vancomycin, daptomycin, and gentamicin confirmed that antibiotic uptake was concentration dependent and that increased porosity correlated with increased uptake and prolonged antibiotic release. We also demonstrate that vancomycin can be passively loaded into either formulation in sufficient concentration to prevent infection in a rabbit model of a contaminated segmental bone defect. Moreover, even in those few cases in which complete eradication was not achieved, the number of viable bacteria in the bone was significantly reduced by treatment and there was no radiographic evidence of osteomyelitis. Radiographs and microcomputed tomography (µCT) analysis from the in vivo studies also suggested that the addition of vancomycin did not have any significant effect on the scaffold itself. These results demonstrate the potential utility of our bone regeneration scaffold for local antibiotic delivery to prevent infection in contaminated bone defects.

We previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in Staphylococcus aureus (Aur, ScpA, SspA/SspB, SplA-F). We also showed that mutation of sarA results in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on in vitro studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo . To this end, we compared the virulence of mgrA , sarA , sarR , sarS , sarZ , and rot mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1, which was isolated directly from the bone of an osteomyelitis patient during surgical debridement. Mutation of mgrA and rot limited virulence to a statistically significant extent in UAMS-1, but not in LAC, while the sarA mutant exhibited reduced virulence in both strains. The reduced virulence of the sarA mutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. These results illustrate the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence and demonstrate the significance of SarA in limiting protease production in vivo in S. aureus .

We previously reported the development of an osteogenic bone filler scaffold consisting of degradable polyurethane (dPU), nano-sized hydroxyapatite (nHA), and decellularized bovine bone particles (DBP). In this report we describe the results of studies aimed at evaluating the use of this scaffold as a means of local antibiotic delivery for the prevention of infection in a segmental bone defect contaminated with Staphylococcus aureus. We evaluated two different scaffold formulations that contained the same components in the same ratios but differed from each other with respect to overall porosity and therefore surface area. Studies done with vancomycin, daptomycin, and gentamicin confirmed that antibiotic uptake was concentration dependent and that increased porosity was correlated with increased uptake and prolonged release of all three antibiotics. Vancomycin could be passively loaded into either scaffold formulation in an amount sufficient to prevent infection, as evidenced by the complete eradication of viable bacteria from the surgical site of most animals in a rabbit model of a contaminated mid-radial segmental bone defect. Even in those few cases in which complete eradication was not achieved, the number of viable bacteria present in the bone was significantly reduced comparison to untreated controls. There was also no radiographic evidence of osteomyelitis in any rabbit treated with vancomycin-loaded scaffold. Microcomputed tomography (μCT) of bone defects up to 84 days of exposure to scaffolds with and without vancomycin also demonstrated that the addition of vancomycin even in the highest concentration did not significantly diminish the osteogenic properties of either scaffold formulation. Together, these results demonstrate the potential utility of our bone regeneration scaffold for local antibiotic delivery.

We previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases inStaphylococcus aureus. We also showed that mutation ofsarAresults in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based onin vitrostudies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteinsin vivo. To this end, we compared the virulence ofmgrA, sarA, sarR, sarS, sarZ, androtmutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1. As assessed based on an overall osteomyelitis pathology score derived from the incidence of bone fracture, bacterial burdens in the bone, cortical bone destruction, and reactive bone formation, mutation ofmgrAandrotlimited virulence to a statistically significant extent in UAMS-1, but not in LAC. In contrast, thesarAmutant exhibited reduced virulence in both strains. This illustrates the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence. The reduced virulence of thesarAmutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. This suggests that thesein vitrophenotypes, either alone or in combination with each other, may be useful in prioritizing additional mutants forin vivoevaluation. Most importantly, they illustrate the significance of limiting protease productionin vivoin S.aureus, and confirm that SarA plays the primary role in this regard.Author SummaryStaphylococcus aureuscauses a diverse array of infections due to its ability to produce an arsenal of virulence factors. Among these are extracellular proteases, which serve several purposes on behalf of the bacterium. However, it has become increasingly apparent that it is also critical to limit the production of these proteases to prevent them from compromising theS. aureusvirulence factor repertoire. Many regulatory loci have been implicated in this respect, but it is difficult to draw relative conclusions because few reports have made direct comparisons, and fewer still have done soin vivo. We addressed this by assessing the impact on virulence of six regulatory loci previously implicated in protease production. We did this in the clinical context of osteomyelitis using mutants generated in two divergent clinical isolates. Our results confirm significant strain-dependent differences, reinforcing the importance of considering such diverse clinical isolates when evaluating targets for potential therapeutic intervention. In this respect, only mutation ofsarAattenuated virulence in both strains. This illustrates the importance of limiting protease production as a means of post-translational regulatory control inS. aureusand confirms thatsarAplays a predominant role in this regard.

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