Increased production of aureolysin and staphopain A is a primary determinant of the reduced virulence of Staphylococcus aureus sarA mutants in osteomyelitis.

We previously demonstrated that mutation of sarA in Staphylococcus aureus limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of these phenotypes can be attributed to the increased production of extracellular proteases. Here we extend these studies to assess the individual importance of these proteases alone and in combination with each other using the methicillin-resistant USA300 strain LAC, the methicillin-susceptible USA200 strain UAMS-1, and isogenic sarA mutants that were also unable to produce aureolysin (Aur), staphopain A (ScpA), staphylococcal serine protease A (subsp.), staphopain B (SspB), and the staphylococcal serine protease-like proteins A-F (SplA-F). Biofilm formation was restored in LAC and UAMS-1 sarA mutants by subsequent mutation of aur and scpA, while mutation of aur had the greatest impact on cytotoxicity to mammalian cells, particularly with conditioned medium (CM) from the more cytotoxic strain LAC. However, SDS-PAGE and western blot analysis of CM confirmed that mutation of sspAB was also required to mimic the phenotype of sarA mutants unable to produce any extracellular proteases. Nevertheless, in a murine model of post-traumatic osteomyelitis, mutation of aur and scpA had the greatest impact on restoring the virulence of LAC and UAMS-1 sarA mutants, with concurrent mutation of sspAB and the spl operon having relatively little effect. These results demonstrate that the increased production of Aur and ScpA in combination with each other is a primary determinant of the reduced virulence of S. aureus sarA mutants in diverse clinical isolates including both methicillin-resistant and methicillin-susceptible strains.IMPORTANCEPrevious work established that SarA plays a primary role in limiting the production of extracellular proteases to prevent them from limiting the abundance of S. aureus virulence factors. Eliminating the production of all 10 extracellular proteases in the methicillin-resistant strain LAC has also been shown to enhance virulence in a murine sepsis model, and this has been attributed to the specific proteases Aur and ScpA. The importance of this work lies in our demonstration that the increased production of these same proteases largely accounts for the decreased virulence of sarA mutants in a murine model of post-traumatic osteomyelitis not only in LAC but also in the methicillin-susceptible human osteomyelitis isolate UAMS-1. This confirms that sarA-mediated repression of Aur and ScpA production plays a critical role in the posttranslational regulation of S. aureus virulence factors in diverse clinical isolates and diverse forms of S. aureus infection.

Comparative evaluation of small molecules reported to be inhibitors of Staphylococcus aureus biofilm formation.

IMPORTANCE: Because biofilm formation is such a problematic feature of Staphylococcus aureus infections, much effort has been put into identifying biofilm inhibitors. However, the results observed with these compounds are often reported in isolation, and the methods used to assess biofilm formation vary between labs, making it impossible to assess relative efficacy and prioritize among these putative inhibitors for further study. The studies we report address this issue by directly comparing putative biofilm inhibitors using a consistent in vitro assay. This assay was previously shown to maximize biofilm formation, and the results observed with this assay have been proven to be relevant in vivo. Of the 19 compounds compared using this method, many had no impact on biofilm formation under these conditions. Indeed, only one proved effective at limiting biofilm formation without also inhibiting growth.

The major role of sarA in limiting Staphylococcus aureus extracellular protease production in vitro is correlated with decreased virulence in diverse clinical isolates in osteomyelitis.

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.

Cartilage-inspired surface textures for improved tribological performance of orthopedic implants.

Joint replacements have become one of the most common orthopedic procedures due to the significant demands of retaining functional mobility. While these procedures are of great value to patients, there are some limitations. Durability is the most important limitation associated with joint replacement that needs to be addressed due to the increasing number of younger patients. Titanium is a commonly used implant material which has high biocompatibility, high strength-to-density ratio, and high corrosion resistance. However, current titanium implants have poor wear resistance which shortens their lifespan. In this study, microscale dimples with four different dimple shapes (circular, triangular, square, and star) of similar sizes to the pores found in natural articular cartilage were fabricated on titanium disks to improve implant lubrication and reduce wear. Biotribology tests were performed on dimpled and non-dimpled titanium disks in a condition similar to that inside of a patient's body. It was shown that dimpling the titanium disks optimized the lubricant film formation and decreased the wear rate significantly while also reducing the coefficient of friction (COF). The star-shaped dimples had the lowest COF and almost no detectable wear after 8 h of testing. To investigate whether dimpling increased bacterial colonization due to increased surface area, and to determine whether any increase could be limited by coating with antibacterial materials, bacterial colonization with Staphylococcus aureus was tested with non-dimpled and star-shaped dimpled titanium disks with and without coating with polydopamine (PDA), silver (Ag) nanoparticles (NPs), and PDA + Ag NPs. It was found that dimpling did not increase bacterial colonization, and that coating with PDA, Ag NPs, or PDA + Ag NPs did not decrease bacterial colonization. Nevertheless, we conclude that star-shaped dimpled titanium surfaces have potential utility as more durable orthopedic implants.

Pyroptosis and pyroptosis-inducing cancer drugs.

Pyroptosis, an inflammatory form of lytic cell death, is a type of cell death mediated by the gasdermin (GSDM) protein family. Upon recognizing exogenous or endogenous signals, cells undergo inflammasome assembly, GSDM cleavage, the release of proinflammatory cytokines and other cellular contents, eventually leading to inflammatory cell death. In this review, we discuss the roles of the GSDM family for anti-cancer functions and various antitumor drugs that could activate the pyroptosis pathways.

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.

As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall's correlation=0.896-0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

Evaluation of a bone filler scaffold for local antibiotic delivery to prevent Staphylococcus aureus infection in a contaminated bone defect.

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.

Evaluation of bone and kidney toxicity of BT2-peg2, a potential carrier for the targeted delivery of antibiotics to bone.

Previous studies have demonstrated that the bone targeting agent BT2-peg2 (BT2-minipeg2, 9), when conjugated to vancomycin and delivered systemically by intravenous (IV) or intraperitoneal (IP) injection accumulates in bone to a greater degree than vancomycin alone, but that this accumulation is associated with severe nephrotoxicity. To determine whether this nephrotoxicity could be attributed to BT2-peg2 itself, we used a rat model to assess the distribution and toxicity of BT2-peg2 after IP injection of 11 mg/kg twice daily for 21 days. The results demonstrated that BT2-peg2 accumulates in bone but there was no evidence of nephrotoxicity or any histopathological abnormalities in the bone. This suggests the nephrotoxicity observed in previous studies is likely due to the altered pharmacokinetics of vancomycin when conjugated to BT2-peg2 rather than to BT2-peg2 itself. Thus, BT2-peg2 may be a safe carrier for the enhanced delivery of antibiotics other than vancomycin to the bone as a means of combating bone infection. However, the data also emphasizes the need to carefully examine the pharmacokinetic characteristics of any BT2-peg2-antibiotic conjugate utilized for treatment of bone infections.

Limiting protease production plays a key role in the pathogenesis of the divergent clinical isolates of Staphylococcus aureus LAC and UAMS-1.

Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.

Staphylococcal infection prevention using antibiotic-loaded mannitol-chitosan paste in a rabbit model of implant-associated osteomyelitis.

Antibiotic-loaded chitosan pastes have shown advantages in the treatment and coverage of complex musculoskeletal defects. We added mannitol, previously shown to increase antibiotic susceptibility of biofilm, to an injectable chitosan/polyethylene glycol paste for delivery of antibiotics. Ground sponges (0.85% acetic acid solution, 1% chitosan, 0% or 2% mannitol, 1% polyethylene glycol) were hydrated using phosphate-buffered saline with 10 mg/ml amikacin and 10 mg/ml vancomycin added to form pastes. We inoculated rabbit radial defects with 105 colony-forming units of Staphylococcus aureus (UAMS-1) and inserted titanium pins into the cortical bone. Groups compared included mannitol blend pastes, non-mannitol blends, antibiotic-loaded bone cement, vancomycin powder, and no treatment controls. We harvested tissue samples and retrieved the pins retrieved at 3 weeks. All antibiotic-loaded groups lowered bacterial growth and colony-forming unit counts in soft and bone tissue and on titanium pins in in vivo studies. The results indicate this biomaterial is capable of eluting active antibiotics at concentrations that reduce bacterial growth on biomaterials and tissue, which, in turn, may prevent biofilm formation. Blends of chitosan and mannitol may be useful in prevention and treatment of osteomyelitis and implant-associated infections.

The Increased Accumulation of Staphylococcus aureus Virulence Factors Is Maximized in a purR Mutant by the Increased Production of SarA and Decreased Production of Extracellular Proteases.

Mutation of purR was previously shown to enhance the virulence of Staphylococcus aureus in a murine sepsis model, and this cannot be fully explained by increased expression of genes within the purine biosynthesis pathway. Rather, the increased production of specific S. aureus virulence factors, including alpha toxin and the fibronectin-binding proteins, was shown to play an important role. Mutation of purR was also shown previously to result in increased abundance of SarA. Here, we demonstrate by transposon sequencing that mutation of purR in the USA300 strain LAC increases fitness in a biofilm while mutation of sarA has the opposite effect. Therefore, we assessed the impact of sarA on reported purR-associated phenotypes by characterizing isogenic purR, sarA, and sarA/purR mutants. The results confirmed that mutation of purR results in increased abundance of alpha toxin, protein A, the fibronectin-binding proteins, and SarA, decreased production of extracellular proteases, an increased capacity to form a biofilm, and increased virulence in an osteomyelitis model. Mutation of sarA had the opposite effects on all of these phenotypes and, other than bacterial burdens in the bone, all of the phenotypes of sarA/purR mutants were comparable to those of sarA mutants. Limiting the production of extracellular proteases reversed all of the phenotypes of sarA mutants and most of those of sarA/purR mutants. We conclude that a critical component defining the virulence of a purR mutant is the enhanced production of SarA, which limits protease production to an extent that promotes the accumulation of critical S. aureus virulence factors.

SarA plays a predominant role in controlling the production of extracellular proteases in the diverse clinical isolates of Staphylococcus aureus LAC and UAMS-1.

Using DNA affinity chromatography we demonstrate that the S. aureus regulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarA and examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarA plays a predominant role in this regard. Using sarA mutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarA mutants, while aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.

Real-Time Monitoring of Bacteria Clearance From Blood in a Murine Model.

Bloodstream infections, especially those that are antibiotic resistant, pose a significant challenge to health care leading to increased hospitalization time and patient mortality. There are different facets to this problem that make these diseases difficult to treat, such as the difficulty to detect bacteria in the blood and the poorly understood mechanism of bacterial invasion into and out of the circulatory system. However, little progress has been made in developing techniques to study bacteria dynamics in the bloodstream. Here, we present a new approach using an in vivo flow cytometry platform for real-time, noninvasive, label-free, and quantitative monitoring of the lifespan of green fluorescent protein-expressing Staphylococcus aureus and Pseudomonas aeruginosa in a murine model. We report a relatively fast average rate of clearance for S. aureus (k = 0.37 ± 0.09 min-1 , half-life ~1.9 min) and a slower rate for P. aeruginosa (k = 0.07 ± 0.02 min-1 , half-life ~9.6 min). We also observed what appears to be two stages of clearance for S. aureus, while P. aeruginosa appeared only to have a single stage of clearance. Our results demonstrate that an advanced research tool can be used for studying the dynamics of bacteria cells directly in the bloodstream, providing insight into the progression of infectious diseases in circulation. © 2019 International Society for Advancement of Cytometry.

The Impacts of msaABCR on sarA-Associated Phenotypes Are Different in Divergent Clinical Isolates of Staphylococcus aureus.

The staphylococcal accessory regulator (sarA) plays an important role in Staphylococcus aureus infections, including osteomyelitis, and the msaABCR operon has been implicated as an important factor in modulating expression of sarA Thus, we investigated the contribution of msaABCR to sarA-associated phenotypes in the S. aureus clinical isolates LAC and UAMS-1. Mutation of msaABCR resulted in reduced production of SarA and a reduced capacity to form a biofilm in both strains. Biofilm formation was enhanced in a LAC msa mutant by restoring the production of SarA, but this was not true in a UAMS-1 msa mutant. Similarly, extracellular protease production was increased in a LAC msa mutant but not a UAMS-1 msa mutant. This difference was reflected in the accumulation and distribution of secreted virulence factors and in the impact of extracellular proteases on biofilm formation in a LAC msa mutant. Most importantly, it was reflected in the relative impact of mutating msa as assessed in a murine osteomyelitis model, which had a significant impact in LAC but not in UAMS-1. In contrast, mutation of sarA had a greater impact on all of these in vitro and in vivo phenotypes than mutation of msaABCR, and it did so in both LAC and UAMS-1. These results suggest that, at least in osteomyelitis, it would be therapeutically preferable to target sarA rather than msaABCR to achieve the desired clinical result, particularly in the context of divergent clinical isolates of S. aureus.

Exploiting Correlations between Protein Abundance and the Functional Status of saeRS and sarA To Identify Virulence Factors of Potential Importance in the Pathogenesis of Staphylococcus aureus Osteomyelitis.

We used a murine model of postsurgical osteomyelitis (OM) to evaluate the relative virulence of the Staphylococcus aureus strain LAC and five isogenic variants that differ in the functional status of saeRS and sarA relative to each other. LAC and a variant in which saeRS activity is increased (saeC) were comparably virulent to each other, while ΔsaeRS, ΔsarA, ΔsaeRS/ΔsarA, and saeC/ΔsarA mutants were all attenuated to a comparable degree. Phenotypic comparisons including a mass-based proteomics approach that allowed us to assess the number and abundance of full-length proteins suggested that mutation of saeRS attenuates virulence in our OM model owing primarily to the decreased production of S. aureus virulence factors, while mutation of sarA does so owing to protease-mediated degradation of these same virulence factors. This was confirmed by demonstrating that eliminating protease production restored virulence to a greater extent in a LAC sarA mutant than in the isogenic saeRS mutant. Irrespective of the mechanism involved, mutation of saeRS or sarA was shown to result in reduced accumulation of virulence factors of potential importance. Thus, using our proteomics approach we correlated the abundance of specific proteins with virulence in these six strains and identified 14 proteins that were present in a significantly increased amount (log2 ≥ 5.0) in both virulent strains by comparison to all four attenuated strains. We examined biofilm formation and virulence in our OM model using a LAC mutant unable to produce one of these 14 proteins, specifically staphylocoagulase. The results confirmed that mutation of coa limits biofilm formation and, to a lesser extent, virulence in our OM model, although in both cases the limitation was reduced by comparison to the isogenic sarA mutant.

Infection of Primary Human Alveolar Macrophages Alters Staphylococcus aureus Toxin Production and Activity.

Pulmonary pathogens encounter numerous insults, including phagocytic cells designed to degrade bacteria, while establishing infection in the human lung. Staphylococcus aureus is a versatile, opportunistic pathogen that can cause severe pneumonia, and methicillin-resistant isolates are of particular concern. Recent reports present conflicting data regarding the ability of S. aureus to survive and replicate within macrophages. However, due to use of multiple strains and macrophage sources, making comparisons between reports remains difficult. Here, we established a disease-relevant platform to study innate interactions between S. aureus and human lungs. Human precision-cut lung slices (hPCLS) were subjected to infection by S. aureus LAC (methicillin-resistant) or UAMS-1 (methicillin-sensitive) isolates. Additionally, primary human alveolar macrophages (hAMs) were infected with S. aureus, and antibacterial activity was assessed. Although both S. aureus isolates survived within hAM phagosomes, neither strain replicated efficiently in these cells. S. aureus was prevalent within the epithelial and interstitial regions of hPCLS, with limited numbers present in a subset of hAMs, suggesting that the pathogen may not target phagocytic cells for intracellular growth during natural pulmonary infection. S. aureus-infected hAMs mounted a robust inflammatory response that reflected natural human disease. S. aureus LAC was significantly more cytotoxic to hAMs than UAMS-1, potentially due to isolate-specific virulence factors. The bicomponent toxin Panton-Valentine leukocidin was not produced during intracellular infection, while alpha-hemolysin was produced but was not hemolytic, suggesting that hAMs alter toxin activity. Overall, this study defined a new disease-relevant infection platform to study S. aureus interaction with human lungs and to define virulence factors that incapacitate pulmonary cells.

Comparative Study of Antibiotic Elution Profiles From Alternative Formulations of Polymethylmethacrylate Bone Cement.

BACKGROUND: Polymethylmethacrylate (PMMA) bone cement is commonly used in orthopedic surgery for implant fixation and local antibiotic delivery following surgical debridement. The incidence of nephrotoxicity necessitates the balance of antiinfective properties with the potential for toxicity. Thus, understanding antibiotic elution characteristics of different PMMA formulations is essential. We sought to address this by assessing elution of vancomycin, daptomycin, and tobramycin from Palacos LV (Palacos), Stryker Surgical Simplex P (Simplex), BIOMET Cobalt HV (Cobalt), and Zimmer Biomet Bone Cement R (Zimmer) radiopaque bone cements. METHODS: Antibiotics were mixed with each cement formulation, and molds were used to produce beads of cement. Beads were incubated in phosphate-buffered saline at 37°C, and antibiotic elution was measured daily for 10 days with vancomycin and 5 days with daptomycin and tobramycin. Active antibiotic was quantified by serial dilution and comparison to the minimum inhibitory concentration. RESULTS: The elution profiles of Simplex were significantly lower than all other cements with all antibiotics (P < .00093). Palacos exhibited a significantly higher vancomycin elution profile than all other cements (P < .00001). The difference in daptomycin elution profiles for Cobalt and Palacos was not significant (P > .43), but both were significantly higher than Zimmer (P < .0006). CONCLUSION: Overall, Stryker Surgical Simplex P exhibits a significantly lower elution profile than all other cements tested. In general, Palacos LV exhibits an increased elution profile compared with other cements. This elution information may assist the surgeon in choosing different cement formulations for the local delivery of antibiotics.

Label-Free Proteomic Approach to Characterize Protease-Dependent and -Independent Effects of sarA Inactivation on the Staphylococcus aureus Exoproteome.

The staphylococcal accessory regulator A ( sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. We previously used a proteomics approach that measures protein abundance of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assesses the impact of this on the accumulation of S. aureus exoproteins. Our previous approach was limited as it did not take into account that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, our goal was to use an expanded proteomics approach utilizing a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels to alleviate these false negatives and thereby provide for characterization of protease-dependent and -independent effects of sarA mutation on the S. aureus exoproteome. Proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases ( sarA/protease) were resolved using one-dimensional gel electrophoresis. Quantitative proteomic comparisons of sarA versus sarA/protease mutants identified proteins that were cleaved in a protease-dependent manner owing to mutation of sarA, and comparisons of sarA/protease mutant versus the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This expanded approach provided for a more comprehensive analysis of the impact of mutating sarA on the S. aureus exoproteome.

Ciprofloxacin and Rifampin Dual Antibiotic-Loaded Biopolymer Chitosan Sponge for Bacterial Inhibition.

Complex extremity wounds in Wounded Warriors can become contaminated with microbes, which may cause clinical outcomes resulting in amputation, morbidity, or even fatality. Local delivery of multiple or broad-spectrum antibiotics allows practicing clinicians treatment solutions that may inhibit biofilm formation. Propagation of vancomycin-resistant Staphylococcus aureus is also a growing concern. The development of vancomycin-resistant S. aureus has become a critical challenge in nosocomial infection prevention in the USA, but to date has seen little occurrence in osteomyelitis. As an alternative, locally delivered ciprofloxacin and rifampin were investigated in a preclinical model for the prevention of biofilm in complex extremity wounds with implanted fixation device. In vitro assays demonstrated ciprofloxacin and rifampin possess an additive effect against Gram-negative Pseudomonas aeruginosa and were actively eluted from a chitosan sponge based local delivery system. In an in vivo orthopedic hardware-associated polymicrobial model (S. aureus and Escherichia coli) the combination was able to achieve complete clearance of both bacterial strains. E. coli was detected in bone of untreated animals, but did not form biofilm on wires. Results reveal the clinical potential of antibiotic-loaded chitosan sponges to inhibit infection through tailored antibiotic selection at desired concentrations with efficacy towards biofilm inhibition.

Local Delivery of Amikacin and Vancomycin from Chitosan Sponges Prevent Polymicrobial Implant-Associated Biofilm.

Military personnel have high risk for infection, particularly those with combat-related extremity trauma. Administration of multiple or broad-spectrum antibiotics provides clinicians with a strategy for preventing biofilm-based medical device infections. Selection of effective antibiotic combinations based on common pathogens may be used to improve chitosan wound dressing sponge-based local antibiotic delivery systems. In vitro assays in this study demonstrate that vancomycin and amikacin have a synergistic relationship against a strain of osteomyelitis-producing Gram-positive Staphylococcus aureus, although an indifferent relationship was observed against Gram-negative Pseudomonas aeruginosa. In an in vivo model of orthopedic hardware-associated polymicrobial (S. aureus and Escherichia coli) biofilm, chitosan sponges loaded with a combination of vancomycin and amikacin at 5 mg/mL each showed a greater percentage of complete clearance, 50%, than either antibiotic alone, 8.33%. Doubling the loading concentration of the combination achieved a complete clearance rate of 100%, a four log-fold reduction of S. aureus on the wire and a six log-fold reduction in bone. E. coli was detected in bone of untreated animals but did not form biofilm on wires. Results demonstrate the clinical potential of chitosan sponges to prevent infection and illustrates antibiotic selection and loading concentrations necessary for effective biofilm prevention.