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.

Evaluation of a chitosan-polyethylene glycol paste as a local antibiotic delivery device.

AIM: To investigate the efficacy of a chitosan/polyethylene glycol blended paste as a local antibiotic delivery device, particularly in musculoskeletal wounds. METHODS: Acidic (A) chitosan sponges and neutralized (N) chitosan/polyethylene glycol (PEG) blended sponges were combined in ratios of 3A:2N, 1A:1N, and 2A:3N; then hydrated with phosphate buffered saline to form a chitosan/PEG paste (CPP). Both in vitro and in vivo studies were conducted to determine the potential CPP has as a local antibiotic delivery device. In vitro biocompatibility was assessed by the cytotoxic response of fibroblast cells exposed to the experimental groups. Degradation rate was measured as the change in dry mass due to lysozyme based degradation over a 10-d period. The antibiotic elution profiles and eluate activity of CPP were evaluated over a 72-h period. To assess the in vivo antimicrobial efficacy of the CPP, antibiotic-loaded paste samples were exposed to subcutaneously implanted murine catheters inoculated with Staphylococcus aureus. Material properties of the experimental paste groups were evaluated by testing the ejection force from a syringe, as well as the adhesion to representative musculoskeletal tissue samples. RESULTS: The highly acidic CPP group, 3A:2N, displayed significantly lower cell viability than the control sponge group. The equally distributed group, 1A:1N, and the highly neutral group, 2A:3N, displayed similar cell viability to the control sponge group and are deemed biocompatible. The degradation studies revealed CPP is more readily degradable than the chitosan sponge control group. The antibiotic activity studies indicated the CPP groups released antibiotics at a constant rate and remained above the minimum inhibitory concentrations of the respective test bacteria for a longer time period than the control chitosan sponges, as well as displaying a minimized burst release. The in vivo functional model resulted in complete bacterial infection prevention in all catheters treated with the antibiotic loaded CPP samples. All experimental paste groups exhibited injectability and adhesive qualities that could be advantageous material properties for drug delivery to musculoskeletal injuries. CONCLUSION: CPP is an injectable, bioadhesive, biodegradable, and biocompatible material with potential to allow variable antibiotic loading and active, local antibiotic release to prevent bacterial contamination.

Trivalent M-related protein as a component of next generation group A streptococcal vaccines.

PURPOSE: There is a need to broaden protective coverage of M protein-based vaccines against group A streptococci (GAS) because coverage of the current 30-valent M protein vaccine does not extend to all emm types. An additional GAS antigen and virulence factor that could potentially extend vaccine coverage is M-related protein (Mrp). Previous work indicated that there are three structurally related families of Mrp (MrpI, MrpII, and MrpIII) and peptides of all three elicited bactericidal antibodies against multiple emm types. The purpose of this study was to determine if a recombinant form containing Mrp from the three families would evoke bactericidal antiserum and to determine if this antiserum could enhance the effectiveness of antisera to the 30-valent M protein vaccine. MATERIALS AND METHODS: A trivalent recombinant Mrp (trMrp) protein containing N-terminal fragments from the three families (trMrp) was constructed, purified and used to immunize rabbits. Anti-trMrp sera contained high titers of antibodies against the trMrp immunogen and recombinant forms representing MrpI, MrpII, and MrpIII. RESULTS: The antisera opsonized emm types of GAS representing each Mrp family and also opsonized emm types not covered by the 30-valent M protein-based vaccine. Importantly, a combination of trMrp and 30-valent M protein antiserum resulted in higher levels of opsonization of GAS than either antiserum alone. CONCLUSION: These findings suggest that trMrp may be an effective addition to future constructs of GAS vaccines.

Blended Chitosan Paste for Infection Prevention: Preliminary and Preclinical Evaluations.

BACKGROUND: Local drug delivery devices offer a promising method for delivering vancomycin and amikacin for musculoskeletal wounds. However, current local delivery devices such as beads and sponges do not necessarily allow for full coverage of a wound surface with eluted antibiotics and do not address the need for reducing the antibiotic diffusion distance to help prevent contamination by bacteria or other microorganisms. We blended chitosan/polyethylene glycol (PEG) pastes/sponges to increase biocompatibility and improve antibiotic coverage within the wound. QUESTIONS/PURPOSES: (1) Are blended chitosan/PEG pastes biodegradable? (2) Are the blended pastes biocompatible? (3) How much force does paste require for placement by injection? (4) Will the pastes elute active antibiotics to inhibit bacteria in vitro? (5) Can the pastes prevent infection in a preclinical model with hardware? METHODS: Our blended paste/sponge formulations (0.5% acidic, 1% acidic, and acidic/neutral) along with a control neutral 1% chitosan sponge were tested in vitro for degradability, cytocompatibility, injectability tested by determining the amount of force needed to inject the pastes, elution of antibiotics, and activity tested using zone of inhibition studies. Along with these studies, in vivo models for biocompatibility and infection prevention were tested using a rodent model and an infected mouse model with hardware, respectively. By evaluating these characteristics, an improved local drug delivery device can be determined. RESULTS: All three of the paste formulations evaluated were almost fully degraded and with 6 days of degradation, the percent remaining being was less than that of the control sponge (percent remaining: control 99.251% ± 1.0%; 0.5% acidic 1.6% ± 2.1%, p = 0.002; 1% acidic 1.7% ± 1.6%, p = 0.002; acidic/neutral 2.3% ± 1.7%, p = 0.010). There was good biocompatibility because cell viability in vitro was high (control 100.0 ± 14.3; 0.5% acidic formulation at 79.4 ± 12.6, p < 0.001; 1% acidic formulation at 98.6 ± 6.1, p = 0.993; acidic/neutral formulation at 106.7 ± 12.8, p = 0.543), and in vivo inflammation was moderate (control 2.1 ± 1.2; 0.5% acidic 3.3 ± 0.2, p = 0.530; 1% acidic 2.5 ± 0.9, p = 0.657; acidic/neutral 2.9 ± 1.1, p = 0.784). Force required to inject the 0.5% acidic and 1% acidic pastes was less than the acidic/neutral paste used as a control (control 167.7 ± 85.6; 0.5% acidic 41.3 ± 10.7, p = 0.070; 1% acidic 28.0 ± 7.0, p = 0.940). At 72 hours, all paste formulations exhibited in vitro activity against Staphylococcus aureus (control 2.6 ± 0.8; 0.5% acidic 98.1 ± 33.5, p = 0.002; 1% acidic 87.3 ± 17.2, p = 0.006; acidic/neutral 83.5 ± 14.3, p = 0.010) and Pseudomonas aeruginosa (control 163.0 ± 1.7; 0.5% acidic 85.7 ± 83.6, p = 0.373; 1% acidic 38.0 ± 45.1, p = 0.896; acidic/neutral 129.7 ± 78.0, p = 0.896). Also, the paste formulations were able to prevent the infection with 100% clearance on the implanted hardware and surrounding tissue with the control being a 0.5% acidic paste group without antibiotics (control 4 × 104 ± 4.8 × 104; 0.5% acidic 0.0 ± 0.0, p value: 0.050; 1% acidic 0.0 ± 0.0, p = 0.050; acidic/neutral 0.0 ± 0.0, p = 0.050). CONCLUSIONS: The preliminary studies demonstrated promising results for the blended chitosan/PEG pastes with antibiotics provided degradability, biocompatibility, injectability, and infection prevention for musculoskeletal-type wounds. CLINICAL RELEVANCE: The preliminary studies with the chitosan paste delivered antibiotics to a contaminated musculoskeletal wound with hardware and prevented infection. More studies in a complex musculoskeletal wound and dosage studies are needed for continued development.

Structure-based design of broadly protective group a streptococcal M protein-based vaccines.

BACKGROUND: A major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new "cluster-based" typing system of 175M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines. METHODS: M protein sequences (AA 16-50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4GAS. RESULTS: The synthetic vaccine rabbit antisera reacted with all 17 E4M peptides and demonstrated bactericidal activity against 15/17 E4GAS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4M peptides. CONCLUSIONS: Comprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS.

Polymicrobial Biofilm Inhibition Effects of Acetate-Buffered Chitosan Sponge Delivery Device.

Polymicrobial biofilm-associated implant infections present a challenging clinical problem. Through modifications of lyophilized chitosan sponges, degradable drug delivery devices for antibiotic solution have been fabricated for prevention and treatment of contaminated musculoskeletal wounds. Elution of amikacin, vancomycin, or a combination of both follows a burst release pattern with vancomycin released above minimum inhibitory concentration for Staphylococcus aureus for 72 h and amikacin released above inhibitory concentrations for Pseudomonas aeruginosa for 3 h. Delivery of a vancomycin, amikacin, or a combination of both reduces biofilm formation on polytetrafluoroethylene catheters in an in vivo model of contamination. Release of dual antibiotics from sponges is more effective at preventing biofilm formation than single-loaded chitosan sponges. Treatment of pre-formed biofilm with high-dose antibiotic release from chitosan sponges shows minimal reduction after 48 h. These results demonstrate infection-preventive efficacy for antibiotic-loaded sponges, as well as the need for modifications in the development of advanced materials to enhance treatment efficacy in removing established biofilm.

Streptococcal serum opacity factor promotes cholesterol ester metabolism and bile acid secretion in vitro and in vivo.

Plasma high density lipoprotein-cholesterol (HDL-C) concentrations negatively correlate with atherosclerotic cardiovascular disease. HDL is thought to have several atheroprotective functions, which are likely distinct from the epidemiological inverse relationship between HDL-C levels and risk. Specifically, strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. The major product of the serum opacity factor (SOF) reaction versus HDL is a cholesteryl ester (CE)-rich microemulsion (CERM), which contains apo E and the CE of ~400,000 HDL particles. Huh7 hepatocytes take up CE faster when delivered as CERM than as HDL, in part via the LDL-receptor (LDLR). Here we compared the final RCT step, hepatic uptake and subsequent intracellular processing to cholesterol and bile salts for radiolabeled HDL-, CERM- and LDL-CE by Huh7 cells and in vivo in C57BL/6J mice. In Huh7 cells, uptake from LDL was greater than from CERM (2-4X) and HDL (5-10X). Halftimes for [(14)C]CE hydrolysis were 3.0±0.2, 4.4±0.6 and 5.4±0.7h respectively for HDL, CERM and LDL-CE. The fraction of sterols secreted as bile acids was ~50% by 8h for all three particles. HDL, CERM and LDL-CE metabolism in mice showed efficient plasma clearance of CERM-CE, liver uptake and metabolism, and secretion as bile acids into the gall bladder. This work supports the therapeutic potential of the SOF reaction, which diverts HDL-CE to the LDLR, thereby increasing hepatic CE uptake, and sterol disposal as bile acids.

Apolipoprotein AI deficiency inhibits serum opacity factor activity against plasma high density lipoprotein via a stabilization mechanism.

The reaction of Streptococcal serum opacity factor (SOF) against plasma high-density lipoproteins (HDL) produces a large cholesteryl ester-rich microemulsion (CERM), a smaller neo HDL that is apolipoprotein (apo) AI-poor, and lipid-free apo AI. SOF is active versus both human and mouse plasma HDL. In vivo injection of SOF into mice reduces plasma cholesterol ∼40% in 3 h while forming the same products observed in vitro, but at different ratios. Previous studies supported the hypothesis that labile apo AI is required for the SOF reaction vs HDL. Here we further tested that hypothesis by studies of SOF against HDL from apo AI-null mice. When injected into apo AI-null mice, SOF reduced plasma cholesterol ∼35% in 3 h. The reaction of SOF vs apo AI-null HDL in vitro produced a CERM and neo HDL, but no lipid-free apo. Moreover, according to the rate of CERM formation, the extent and rate of the SOF reaction versus apo AI-null mouse HDL were less than that against wild-type (WT) mouse HDL. Chaotropic perturbation studies using guanidine hydrochloride showed that apo AI-null HDL was more stable than WT HDL. Human apo AI added to apo AI-null HDL was quantitatively incorporated, giving reconstituted HDL. Both SOF and guanidine hydrochloride displaced apo AI from the reconstituted HDL. These results support the conclusion that apo AI-null HDL is more stable than WT HDL because it lacks apo AI, a labile protein that is readily displaced by physicochemical and biochemical perturbations. Thus, apo AI-null HDL is less SOF-reactive than WT HDL. The properties of apo AI-null HDL can be partially restored to those of WT HDL by the spontaneous incorporation of human apo AI. It remains to be determined what other HDL functions are affected by apo AI deletion.

Protective immunogenicity of group A streptococcal M-related proteins.

Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33 mrp genes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GAS emm types expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing different emm types that expressed an Mrp within the same family but showed no activity against emm types expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines.

Novel Antibiotic-loaded Point-of-care Implant Coating Inhibits Biofilm.

BACKGROUND: Orthopaedic biomaterials are susceptible to biofilm formation. A novel lipid-based material has been developed that may be loaded with antibiotics and applied as an implant coating at point of care. However, this material has not been evaluated for antibiotic elution, biofilm inhibition, or in vivo efficacy. QUESTIONS/PURPOSES: (1) Do antibiotic-loaded coatings inhibit biofilm formation? (2) Is the coating effective in preventing biofilm in vivo? METHODS: Purified phosphatidylcholine was mixed with 25% amikacin or vancomycin or a combination of 12.5% of both. A 7-day elution study for coated titanium and stainless steel coupons was followed by turbidity and zone of inhibition assays against Staphylococcus aureus and Pseudomonas aeruginosa. Coupons were inoculated with bacteria and incubated 24 hours (N = 4 for each test group). Microscopic images of biofilm were obtained. After washing and vortexing, attached bacteria were counted. A mouse biofilm model was modified to include coated and uncoated stainless steel wires inserted into the lumens of catheters inoculated with a mixture of S aureus or P aeruginosa. Colony-forming unit counts (N = 10) and scanning electron microscopy imaging of implants were used to determine antimicrobial activity. RESULTS: Active antibiotics with colony inhibition effects were eluted for up to 6 days. Antibiotic-loaded coatings inhibited biofilm formation on in vitro coupons (log-fold reductions of 4.3 ± 0.4 in S aureus and 3.1 ± 0 for P aeruginosa in phosphatidylcholine-only coatings, 5.6 ± 0 for S aureus and 3.1 ± 0 for P aeruginosa for combination-loaded coatings, 5.5 ± 0.3 for S aureus in vancomycin-loaded coatings, and 3.1 ± 0 for P aeruginosa for amikacin-loaded coatings (p < 0.001 for all comparisons of antibiotic-loaded coatings against uncoated controls for both bacterial strains, p < 0.001 for comparison of antibiotic-loaded coatings against phosphatidylcholine only for S aureus, p = 0.54 for comparison of vancomycin versus combination coating in S aureus, P = 0.99 for comparison of antibiotic- and unloaded phosphatidylcholine coatings in P aeruginosa). Similarly, antibiotic-loaded coatings reduced attachment of bacteria to wires in vivo (log-fold reduction of 2.54 ± 0; p < 0.001 for S aureus and 0.83 ± 0.3; p = 0.112 for P aeruginosa). CONCLUSIONS: Coatings deliver active antibiotics locally to inhibit biofilm formation and bacterial growth in vivo. Future evaluations will include orthopaedic preclinical models to confirm therapeutic efficacy. CLINICAL RELEVANCE: Clinical applications of local drug delivery coating could reduce the rate of implant-associated infections.

Identification of a group B streptococcal fibronectin binding protein, SfbA, that contributes to invasion of brain endothelium and development of meningitis.

Group B Streptococcus (GBS) is currently the leading cause of neonatal meningitis. This is due to its ability to survive and multiply in the bloodstream and interact with specialized human brain microvascular endothelial cells (hBMEC), which constitute the blood-brain barrier (BBB). The exact mechanism(s) of GBS-BBB penetration is still largely unknown. We and others have shown that GBS interacts with components of the extracellular matrix. In this study, we demonstrate that GBS of representative serotypes binds immobilized and cell surface fibronectin and identify a putative fibronectin binding protein, streptococcal fibronectin binding protein A (SfbA). Allelic replacement of sfbA in the GBS chromosome resulted in a significant decrease in ability to bind fibronection and invade hBMEC compared with the wild-type (WT) parental strain. Expression of SfbA in the noninvasive strain Lactococcus lactis was sufficient to promote fibronectin binding and hBMEC invasion. Furthermore, the addition of an antifibronectin antibody or an RGD peptide that blocks fibronectin binding to integrins significantly reduced invasion of the WT but not the sfbA-deficient mutant strain, demonstrating the importance of an SfbA-fibronectin-integrin interaction for GBS cellular invasion. Using a murine model of GBS meningitis, we also observed that WT GBS penetrated the brain and established meningitis more frequently than did the ΔsfbA mutant strain. Our data suggest that GBS SfbA plays an important role in bacterial interaction with BBB endothelium and the pathogenesis of streptococcal meningitis.

Composite chitosan and calcium sulfate scaffold for dual delivery of vancomycin and recombinant human bone morphogenetic protein-2.

A biodegradable, composite bone graft, composed of chitosan microspheres embedded in calcium sulfate, was evaluated in vitro for point-of-care loading and delivery of antibiotics and growth factors to prevent infection and stimulate healing in large bone injuries. Microspheres were loaded with rhBMP-2 or vancomycin prior to mixing into calcium sulfate loaded with vancomycin. Composites were evaluated for set time, drug release kinetics, and bacteriostatic/bactericidal activity of released vancomycin, induction of ALP expression by released rhBMP-2, and interaction of drugs on cells. Results showed the composite set in under 36 min and released vancomycin levels that were bactericidal to S. aureus (>MIC 8-16 µg/mL) for 18 days. Composites exhibited a 1 day-delayed release, followed by a continuous release of rhBMP-2 over 6 weeks; ranging from 0.06 to 1.49 ng/mL, and showed a dose dependent release based on initial loading. Released rhBMP-2 levels were, however, too low to induce detectable levels of ALP in W20-17 cells, due to the affinity of rhBMP-2 for calcium-based materials. With stimulating amounts of rhBMP-2 (>50 ng/mL), the ALP response from W-20-17 cells was inhibited when exposed to high vancomycin levels (1,800-3,600 µg/mL). This dual-delivery system is an attractive alternative to single delivery or preloaded systems for bone regeneration since it can simultaneously fight infection and deliver a potent growth factor. Additionally, this composite can accommodate a wide range of therapeutics and thus be customizable for specific patient needs, however, the potential interactive effects of multiple agents must be investigated to ensure that functional activity is not altered.

Non-immune binding of human IgG to M-related proteins confers resistance to phagocytosis of group A streptococci in blood.

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG1>IgG4>IgG2>IgG3. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.

Chitosan sponges for local synergistic infection therapy: a pilot study.

BACKGROUND: Although bacterial antibiotic resistance is increasing, fewer new antibiotics are being developed to compensate. Localized delivery of synergistic antiseptics and antibiotics with a chitosan sponge device may offer an alternative infection treatment. QUESTIONS/PURPOSES: In this pilot study, we asked whether antiseptic and antibiotic combinations provided in vitro synergism against Staphylococcus aureus, whether synergism reduces cell viability, and whether their combination releases drugs at inhibitory levels. METHODS: To investigate the pharmacodynamics among three combinations of the antiseptic chlorhexidine digluconate (CHX) with the antibiotics amikacin, daptomycin, and vancomycin (VAN) (n=1), we determined the fractional inhibitory concentration (FIC) index against S aureus Cowan I. The determined synergistic combination of CHX and VAN was evaluated for cell compatibility using NIH/3T3 fibroblasts (n=3) and the drug release profile from a chitosan sponge device (n=5). RESULTS: With an FIC index<0.5, the combination of CHX+VAN exhibited synergism against S aureus. CHX concentrations≥3.91 µg/mL resulted in fibroblast viability decrease, whereas the combination of CHX+VAN did not decrease fibroblast viability until their concentrations reached ≥7.81 µg/mL. The CHX and VAN release profile, both individually and in combination, was an initial bolus with no difference between eluate concentrations after Day 5. CONCLUSIONS: CHX+VAN combination may be delivered locally by a chitosan sponge that synergistically inhibits S aureus growth. CLINICAL RELEVANCE: The use of synergism between combined antibiotic and antiseptics delivered at high local concentrations with an implanted chitosan sponge may provide a useful alternative infection treatment option.

A daptomycin-xylitol-loaded polymethylmethacrylate bone cement: how much xylitol should be used?

BACKGROUND: The rate of release of an antibiotic from an antibiotic-loaded polymethylmethacrylate (PMMA) bone cement is low. This may be increased by adding a particulate poragen (eg, xylitol) to the cement powder. However, the appropriate poragen amount is unclear. QUESTIONS/PURPOSES: We explored the appropriate amount of xylitol to use in a PMMA bone cement loaded with daptomycin and xylitol. METHODS: We prepared four groups of cement, each comprising the same amount of daptomycin in the powder (1.36 g/40 g dry powder) but different amounts of xylitol (0, 0.7, 1.4, and 2.7 g); the xylitol mass ratio (X) (mass divided by mass of the final dry cement-daptomycin-xylitol mixture) ranged from 0 to 6.13 wt/wt%. Eight mechanical, antibiotic release, and bacterial inhibitory properties were determined using three to 22 specimens or replicates per test. We then used an optimization method to determine an appropriate value of X by (1) identifying the best-fit relationship between the value of each property and X, (2) defining a master objective function incorporating all of the best fits; and (3) determining the value of X at the maximum master objective function. RESULTS: We found an appropriate xylitol amount to be 4.46 wt/wt% (equivalent to 1.93 g xylitol mixed with 1.36 g daptomycin and 40 g dry cement powder). CONCLUSIONS: We demonstrated a method that may be used to determine an appropriate xylitol amount for a daptomycin-xylitol-loaded PMMA bone cement. These findings will require in vivo confirmation. CLINICAL RELEVANCE: While we identified an appropriate amount of xylitol in a daptomycin-xylitol-loaded PMMA bone cement as a prophylactic agent in total joint arthroplasties, clinical evaluations are needed to confirm the effectiveness of this cement.

Preliminary investigation of crosslinked chitosan sponges for tailorable drug delivery and infection control.

Local versus systemic antibiotic delivery may be an effective strategy for treating musculoskeletal infections, especially when antibiotic-resistant bacteria are present. Lyophilized uncrosslinked, genipin crosslinked, and genipin crosslinked with poly(N-isopropylacrylamide) (PNIPAM) chitosan sponges were analyzed for their in vitro degradation rate, chemical crosslinking, antibiotic uptake, elution, biologic activity, and cytotoxicity. These evaluations were pursued to determine if crosslinking with genipin could be used to create a tailorable point of care loaded sponge for local infection control. Crosslinking the chitosan sponges decreased degradation in phosphate-buffered saline from 4.48 ± 2.28 wt % remaining of the uncrosslinked sponges to 78.82 ± 1.15 and 73.87 ± 1.27 wt % remaining at week 1 for the genipin and PNIPAM/genipin crosslinked sponges, respectively. The PNIPAM/genipin crosslinked sponges exhibited the most sustained release of biologically active antibiotics, with an average antibiotic release 63% higher than uncrosslinked and 37% higher than genipin crosslinked sponges, after 96 h. No significant cytotoxic effects from sponges or eluates were exhibited with NIH 3T3 fibroblasts. These preliminary results indicate that genipin crosslinked chitosan sponges, with or without PNIPAM, have potential as local delivery systems for adjunctive therapy for infection control, especially when longer degradation periods and higher antibiotic elutions are desired.

Cis-2-decenoic acid inhibits S. aureus growth and biofilm in vitro: a pilot study.

BACKGROUND: Cis-2 decenoic acid (C2DA) disperses biofilm in many strains of microorganisms. However, whether C2DA inhibits bacterial growth or has potential to boost the actions of antibiotics is unknown. QUESTIONS/PURPOSES: We asked whether (1) C2DA inhibited MRSA growth and biofilm, (2) antibiotics increased inhibitory effects, (3) inhibitory concentrations of C2DA were cytotoxic to human cells, and (4) effective concentrations could be delivered from a chitosan sponge drug delivery device. METHODS: Broth containing seven concentrations of C2DA and six concentrations of either daptomycin, vancomycin, or linezolid was inoculated with a clinical isolate of MRSA and added to a total of 504 coated microtiter plate wells in triplicate (n = 3) for turbidity bacterial growth and crystal violet biofilm mass quantification. We used fibroblast cell viability assays of six C2DA concentrations (n = 4) to evaluate preliminary biocompatibility. We measured the elution of C2DA from a chitosan sponge drug delivery device with two representative loading concentrations (n = 3). RESULTS: C2DA at concentrations of 500 µg/mL and above inhibited growth, while 125 µg/mL C2DA inhibited biofilm. Combination with antibiotics increased these effects. At concentrations up to 500 µg/mL, there were no cytotoxic effects on fibroblasts. Chitosan sponges loaded with 100 mg of C2DA eluted concentrations at or above biofilm-inhibitory concentrations for 5 days. CONCLUSIONS: C2DA inhibited biofilm formation by MRSA at biocompatible concentrations, with increasing biofilm reduction with added antibiotics. Elution of C2DA from a chitosan sponge can be modified through adjusting loading concentration. CLINICAL RELEVANCE: By inhibiting biofilm formation on implant surfaces, C2DA may reduce the number of infections in musculoskeletal trauma.

Apolipoprotein E mediates enhanced plasma high-density lipoprotein cholesterol clearance by low-dose streptococcal serum opacity factor via hepatic low-density lipoprotein receptors in vivo.

OBJECTIVE: Recombinant streptococcal serum opacity factor (rSOF) mediates the in vitro disassembly of human plasma high-density lipoprotein (HDL) into lipid-free apolipoprotein (apo) A-I, a neo-HDL that is cholesterol poor, and a cholesteryl ester-rich microemulsion (CERM) containing apoE. Given the occurrence of apoE on the CERM, we tested the hypothesis that rSOF injection into mice would reduce total plasma cholesterol clearance via apoE-dependent hepatic low-density lipoprotein receptors (LDLR). METHODS AND RESULTS: rSOF (4 µg) injection into wild-type C57BL/6J mice formed neo-HDL, CERM, and lipid-free apoA-I, as observed in vitro, and reduced plasma total cholesterol (-43%, t(1/2)=44±18 minutes) whereas control saline injections had a negligible effect. Similar experiments with apoE(-/-) and LDLR(-/-) mice reduced plasma total cholesterol ≈0% and 20%, respectively. rSOF was potent; injection of 0.18 µg of rSOF produced 50% of maximum reduction of plasma cholesterol 3 hours postinjection, corresponding to a ≈0.5-mg human dose. Most cholesterol was cleared hepatically (>99%), with rSOF treatment increasing clearance by 65%. CONCLUSIONS: rSOF injection into mice formed a CERM that was cleared via hepatic LDLR that recognize apoE. This reaction could provide an alternative mechanism for reverse cholesterol transport.

Evaluation of two sources of calcium sulfate for a local drug delivery system: a pilot study.

BACKGROUND: Local drug delivery has substantial potential to prevent infections compared with systemic delivery. Although calcium sulfate (CaSO(4)) has been studied for local drug delivery and two types are commercially available, it is unknown whether they differentially release antibiotics. QUESTIONS/PURPOSES: We determined the differences between two sources of CaSO(4) and the K(2)SO(4) catalyst's presence on the degradation, daptomycin elution, and activity against Staphylococcus aureus. METHODS: We formed pellets from synthetic and naturally sourced (from gypsum) CaSO(4) and loaded with 5% daptomycin and 3% or 0% K(2)SO(4). We used in vitro experiments to determine the daptomycin concentration and degradation profiles over 10 days. Turbidity assays were used to evaluate the activity of the daptomycin eluates against S. aureus. RESULTS: All pellets exhibited a bolus release with the highest daptomycin concentration on Day 1 with the sourced CaSO(4) pellets. The synthetic CaSO(4) pellets with 3% K(2)SO(4) exhibited a slower drug release compared with the synthetic CaSO(4) pellets with 0% K(2)SO(4), which degraded and eluted daptomycin too quickly to inhibit S. aureus. Turbidity assays demonstrated that all CaSO(4) pellets inhibit S. aureus for expected lengths of time. CONCLUSIONS: Our preliminary in vitro data suggest differences in the degradation, elution, and activity properties between sourced and synthetic CaSO(4) pellets. The addition of K(2)SO(4) appeared beneficial when using synthetic CaSO(4). Synthetic CaSO(4) may be effective when slow degradation and longer elution times are needed. CLINICAL RELEVANCE: Local delivery of eluted daptomycin can be tailored through material selection and K(2)SO(4) addition.

Chitosan coatings deliver antimicrobials from titanium implants: a preliminary study.

OBJECTIVE: Chitosan was investigated as a coating for local delivery of antimicrobials for prevention of acute implant infection. The objectives of this study were to (1) measure the release of 2 antimicrobials from chitosan coatings, (2) determine efficacy of eluted antimicrobials against bacteria, in vitro, and (3) evaluate toxicity of eluted drugs to host cells/tissues. METHODS: Chitosan coatings (80.7% deacetylated, 108 kDa) containing 20% tetracycline or 0.02% chlorhexidine digluconate were bonded to titanium via silane reactions. After elution in culture medium for 7 days, eluates were tested against model pathogens Actinobacillus actinomycetemcomitans and Staphylococcus epidermidis in turbidity tests and in 24-hour cytotoxicity tests using human osteoblasts and fibroblasts. Finally, antibiotic-loaded chitosan-coated titanium pins were implanted for 7 days in muscle of Sprague-Dawley rats to evaluate the initial tissue response. RESULTS: Coatings released 89% of tetracycline in 7 days and 100% chlorhexidine in 2 days. Released tetracycline inhibited growth (95%-99.9%) of pathogens for up to 7 days with no cytotoxicity to human cells. Released chlorhexidine was active against pathogens for 1 to 2 days (56%-99.5% inhibition) but was toxic to cells on the first day of elution. Typical acute inflammatory response was observed to antimicrobial-loaded chitosan coatings similar to unloaded coatings. CONCLUSION: These preliminary data support the hypothesis that chitosan coatings have the potential to locally deliver antimicrobials to inhibit bacteria without being toxic to host cells/tissues and warrant additional studies to evaluate the ability of the coatings to prevent/resist infection and promote osseointegration.