ABSTRACT
Staphylococcus aureus osteomyelitis is a debilitating infection of bone. Treatment of osteomyelitis is impaired by the propensity of invading bacteria to induce pathological bone remodeling that may limit antibiotic penetration to the infectious focus. The nonsteroidal anti-inflammatory drug diflunisal was previously identified as an osteoprotective adjunctive therapy for osteomyelitis, based on the ability of this compound to inhibit S. aureus quorum sensing and subsequent quorum-dependent toxin production. When delivered locally during experimental osteomyelitis, diflunisal significantly limits bone destruction without affecting bacterial burdens. However, because diflunisal’s “quorum-quenching” activity could theoretically increase antibiotic recalcitrance, it is critically important to evaluate this adjunctive therapy in the context of standard-of-care antibiotics. The objective of this study is to evaluate the efficacy of vancomycin to treat osteomyelitis during local diflunisal treatment. We first determined that systemic vancomycin effectively reduces bacterial burdens in a murine model of osteomyelitis and identified a dosing regimen that decreases bacterial burdens without eradicating infection. Using this dosing scheme, we found that vancomycin activity is unaffected by the presence of diflunisal in vitro and in vivo. Similarly, locally delivered diflunisal still potently inhibits osteoblast cytotoxicity in vitro and bone destruction in vivo in the presence of subtherapeutic vancomycin. However, we also found that the resorbable polyester urethane (PUR) foams used to deliver diflunisal serve as a nidus for infection. Taken together, these data demonstrate that diflunisal does not significantly impact standard-of-care antibiotic therapy for S. aureus osteomyelitis, but they also highlight potential pitfalls encountered with local drug delivery.
INTRODUCTION
Osteomyelitis, or inflammation of bone, is most commonly caused by bacterial infection, with Staphylococcus aureus being the most common etiologic agent (1). Acute osteomyelitis can often be successfully treated with a combination of surgical debridement and long courses of antibiotics. In contrast, treatment failure is commonly observed with chronic osteomyelitis, often correlating with the presence of sequestra, which are devitalized fragments of bone that serve as a nidus for persistent infection (1, 2). Furthermore, complications such as pathological fracture and limb-length discrepancy further increase the morbidity of osteomyelitis and may lead to lifelong disability (3, 4). A common underlying factor in the therapeutic recalcitrance and comorbid sequelae of osteomyelitis is dysregulated bone remodeling, including bone destruction that is triggered by both the infecting pathogen and the resultant antibacterial immune response. Taken together, these observations outline the need for adjunctive therapies that protect bone during osteomyelitis, thereby limiting the progression to chronic infection.
We recently used a murine model of staphylococcal osteomyelitis to confirm the seminal findings of Gillaspy et al., who discovered a key role for the S. aureus quorum sensing accessory gene regulator (agr) locus in mediating bone destruction during experimental osteomyelitis in rabbits (5, 6). Based on these findings, we then tested the efficacy of local delivery of the nonsteroidal anti-inflammatory drug (NSAID) diflunisal in the murine osteomyelitis model (7). Diflunisal is a salicylic acid derivative that was identified in an in silico screen for compounds that might inhibit histidine kinase (AgrC)-mediated phosphorylation of the response regulator AgrA, and was found to mitigate virulence factor production without affecting bacterial growth (8). Consistent with these findings, we found that diflunisal potently inhibited Agr-mediated cytotoxicity toward osteoblasts (7). Moreover, local delivery of diflunisal in vivo in a murine model of S. aureus osteomyelitis decreased cortical bone destruction independently of changes in bacterial burden. Collectively, these findings outlined the feasibility of diflunisal as an adjunctive therapy for staphylococcal osteomyelitis, which adds to a growing body of literature suggesting that so-called “quorum quenching” might be an effective strategy to limit staphylococcal virulence during invasive infection (9–13).
Although quorum-quenching approaches have shown great preclinical promise for the treatment of staphylococcal disease, functional suppression of the Agr pathway could also have detrimental effects on antimicrobial therapy for S. aureus. Inactivation of the agr locus leads to enhanced biofilm formation in vitro, which could conceivably limit antibiotic diffusion into the infectious niche (14–16). Moreover, clinical isolates obtained from patients suffering from chronic, invasive staphylococcal infections, including osteomyelitis, frequently have inactivating mutations in the agr locus (17–20). Thus, functional suppression of Agr-mediated gene regulation may represent an adaptation to host tissues during chronic infection. These observations suggest that quorum-quenching agents could potentially exacerbate staphylococcal disease, and therefore, these compounds should be evaluated in the context of standard-of-care antibiotic therapy.
The objective of this study was to evaluate the efficacy of the quorum-quenching drug diflunisal in the context of systemic antibiotic therapy during S. aureus osteomyelitis. We hypothesized that adjunctive diflunisal therapy would inhibit S. aureus-mediated bone destruction and would not interfere with systemic antibiotic activity at the infectious focus. However, we also entertained the hypothesis that diflunisal would decrease antibiotic efficacy, resulting in an infection that is more recalcitrant to standard-of-care antibiotic therapy. To test these hypotheses, we evaluated vancomycin treatment of S. aureus in the presence and absence of diflunisal, both in vitro and in vivo.
RESULTS
Porosity and drug release of diflunisal-loaded PUR foams.Scanning electron microscopy (SEM) was performed to determine the effects of diflunisal loading on porosity and pore size of polyester urethane (PUR) foams (Fig. 1A to C). Empty foams exhibited 89 ± 2% porosity (Fig. 1D) and pore diameter 190 ± 150 μm (Fig. 1E), consistent with previous studies (21–23). PUR foams loaded with 10 mM diflunisal demonstrated 85 ± 7% porosity and pore diameter 135 ± 130 μm, and PUR foams containing 20 mM diflunisal had 92 ± 3% porosity and pore diameter 190 ± 172 μm. To characterize diflunisal release, diflunisal-loaded PUR foams were incubated in phosphate-buffered saline (PBS) at 37°C for 14 days, and diflunisal concentration released from the foams was analyzed using a UV-visible (UV-vis) plate reader. Diflunisal has an amphiphilic profile in water with a solubility of 14.5 mg/liter and an octanol/water partition coefficient of 4.4. We therefore anticipated a slow release of diflunisal from the PUR foam (24). A bolus release of 5% (∼8 μg) was observed on day 1, and ∼20% of the total diflunisal in the PUR foam was released after 14 days (Fig. 1F).
Characterization of PUR and diflunisal-loaded PUR. SEM images of PUR foams containing 0 (A), 10 (B), and 20 mM (C) diflunisal. Scale bar, 200 μm. SEM images of PUR foams were used to determine porosity (%) (D) and pore diameter (μm) (E). (F) Release of diflunisal from PUR. Cumulative release was quantified as the amount of diflunisal released in the leachate normalized by the original amount of diflunisal in the foams. Daily release was quantified as the amount of diflunisal in the leachate at each time point. Error bars represent standard deviation (SD). n = 6 per group.
Diflunisal does not inhibit vancomycin activity in vitro.Functional inactivation of the Agr system could theoretically decrease antibiotic efficacy through altered bacterial metabolism. We therefore investigated if quorum-quenching therapy with diflunisal inhibited the effectiveness of vancomycin in vitro. To investigate the effects of simultaneous delivery of vancomycin and diflunisal, both compounds were delivered in a solubilized form to a subculture of S. aureus. Vancomycin concentrations both above and below the MIC (1 μg/ml) were chosen for these experiments. Soluble diflunisal was delivered at 25 μg/ml, a concentration comparable to the total payload delivered by a diflunisal-loaded foam. Enumeration of CFUs was performed 24 h after simultaneous vancomycin and diflunisal therapy (Fig. 2A). To determine how pretreatment of S. aureus with diflunisal might influence subsequent susceptibility to vancomycin, bacteria were cultured in the presence (25 μg/ml) or absence of diflunisal at 37°C with shaking for 15 h. Bacteria were then subcultured into fresh media containing diflunisal at 25 μg/ml and vancomycin at concentrations above and below the MIC (Fig. 2B). Vancomycin remained effective in the presence of diflunisal cotreatment, both with simultaneous treatment and with pretreatment of bacteria prior to vancomycin. Collectively, these results indicate that diflunisal does not significantly impact the antibacterial activity of vancomycin in vitro.
Diflunisal does not inhibit vancomycin activity against S. aureus in vitro. S. aureus was cultured overnight with (A) or without (B) 25 μg/ml diflunisal. Diflunisal concentration was based on the total amount of diflunisal released from the foams as determined in Fig. 1. Vancomycin was delivered at concentrations near the MIC with and without diflunisal to characterize the effect of combined delivery on the bactericidal capability of vancomycin. n = 3 technical replicates per group. Error bars represent SD. Data are representative of 3 independent trials. Significance determined by Student's t test.
Diflunisal released from PUR foams inhibits staphylococcal cytotoxicity in vitro.Avascular surfaces are readily colonized by bacteria at an infected site, particularly during bone grafting (25, 26). Therefore, in order to minimize the foreign body surface for bacterial colonization, PUR foam sizes were decreased compared to our previous study (7). To accommodate a smaller PUR foam and ensure a similar payload was delivered, PUR foams containing 0 mM, 10 mM (the concentration of diflunisal previously tested [7]), and 20 mM (similar payload to previously tested foams) diflunisal were synthesized. S. aureus cultures were grown in the presence of PUR foams containing each concentration of diflunisal, after which concentrated supernatants were prepared and tested for osteoblast cytotoxicity. Overnight cultures exhibited similar CFU concentrations, suggesting diflunisal-loaded PUR did not affect bacterial growth (Fig. S1A in the supplemental material). Diflunisal-loaded foams dose-dependently inhibited supernatant toxicity toward MC3T3 cells (Fig. S1B). Based on these results, PUR with 20-mM diflunisal foams were chosen for subsequent in vivo studies to minimize foam size while maintaining optimal diflunisal payload.
Vancomycin does not inhibit diflunisal activity in vitro.Diflunisal dose-dependently inhibits S. aureus-induced osteoblast cell death (7). Although we did not expect vancomycin to affect diflunisal activity, we prepared S. aureus supernatants grown in the presence of diflunisal or diflunisal-vancomycin for subsequent MC3T3 treatment. A concentration of 0.1 μg/ml vancomycin (below the vancomycin MIC) was chosen to ensure bacterial growth yields remained comparable. Overnight cultures containing vehicle control (dimethyl sulfoxide [DMSO]), diflunisal, and diflunisal-vancomycin exhibited similar CFU concentrations (Fig. S2A). Treatment with diflunisal-vancomycin significantly limited the cytotoxicity of prepared supernatants compared to the vehicle control group (Fig. S2B). Furthermore, diflunisal-vancomycin treatment was not significantly different than diflunisal treatment. Thus, vancomycin did not affect diflunisal activity in vitro.
Subcutaneous vancomycin reduces bacterial burdens in vivo during osteomyelitis.Prior to evaluating the impact of local diflunisal therapy on antimicrobial activity of systemically administered vancomycin, we first sought to establish an effective subcutaneous dosing regimen for vancomycin that decreased bacterial burdens without completely eradicating infection. To accomplish this, groups of mice (n = 5) were intraosseously infected with 106 CFU of S. aureus, and vancomycin treatment was started immediately after infection via subcutaneous injection of 0, 10, 20, or 30 mg/kg every 12 h. At 7 days postinfection, bacterial burdens were enumerated from the infected femur. Systemic vancomycin treatment at a dose of 30 mg/kg every 12 h nearly eradicated the infection, with 2 mice exhibiting no detectable CFU in the infected femur and the remaining 3 mice exhibiting bacterial burdens near the limit of detection. Decreasing the vancomycin dose resulted in a stepwise increase in bacterial burdens, with the 10-mg/kg dose resulting in an approximately 1-log reduction (P < 0.05) in bacterial burdens (Fig. 3A). This dose was chosen for subsequent experiments testing the impact of local diflunisal therapy. Together, these data reveal that subcutaneous vancomycin therapy is effective for control of experimental S. aureus osteomyelitis when given immediately following infection, and they identify an appropriate subtherapeutic dose for testing additional adjunctive therapies.
Local diflunisal therapy does not inhibit systemic vancomycin activity in vivo. (A) Vancomycin was delivered at 4 different concentrations to determine a suboptimal dose that would significantly decrease the bacterial burdens in murine femurs infected with S. aureus (n = 5 mice per group). Horizontal lines represent the mean. Error bars represent SD. Dotted line depicts the limit of detection (LOD) for bacterial burdens. *, P < 0.05; ****, P < 0.0001. (B, C) Mice were treated with either PUR+Veh or PUR+Dif (see Table 1). At 14 days postinfection, femurs (B) and foams (C) were harvested for CFU enumeration. n = 4 or 5 mice per group (one mouse in the PUR+Veh group had to be euthanized according to humane endpoints). Control group demonstrates bacterial burdens consistent with separate trials (data not shown). *, P < 0.05 relative to PUR+Veh treatment as determined by Student's t test.
Local diflunisal delivery does not improve vancomycin-mediated bacterial clearance in bone.Although we have previously demonstrated that local diflunisal treatment can decrease pathogen-induced cortical bone destruction in a murine osteomyelitis model (7), the efficacy of diflunisal in the setting of concurrent systemic antibiotic therapy is unknown. To test the impact of local diflunisal treatment on the antimicrobial efficacy of systemic vancomycin therapy, groups of mice (n = 5) were subjected to experimental osteomyelitis and systemic vancomycin therapy at 10 mg/kg every 12 h. Diflunisal was delivered locally by elution from a PUR foam (PUR+Dif) as previously reported (7). An empty PUR foam served as a control for local therapy. Bacterial burdens were determined at day 7 postinfection by homogenizing the infected femur as well as residual PUR foam. Systemic vancomycin (Vanc) therapy was effective in decreasing bacterial burdens in the femurs from PUR+Vanc and PUR+Dif+Vanc mice (Fig. 3B). No additive effect was observed with combined vancomycin and diflunisal therapy in terms of decreasing bacterial burdens. Although systemic vancomycin therapy effectively reduced bacterial burdens in bone tissue, it failed to reduce bacterial burdens present on the surface of both PUR and PUR+Dif foams (Fig. 3C), suggesting that the PUR foams are serving as a nidus of infection at the surgical site. Finally, because diflunisal inhibits Agr-mediated quorum sensing and therefore may impact dissemination of infection, we compared bacterial burdens in the kidneys and livers of infected mice. Although infection had disseminated to the kidneys and livers of some mice, no significant difference was observed between PUR+vehicle control (Veh) and PUR+Dif+Vanc, groups suggesting that the addition of diflunisal or vancomycin had no appreciable effect on bacterial dissemination from the femur (Fig. S3). Taken together, these data reveal that local diflunisal therapy does not impede the antimicrobial actions of systemic vancomycin therapy. However, local diflunisal delivery did not improve antibiotic killing by vancomycin under these experimental conditions. Moreover, the resorbable PUR foam, which was originally designed to function as an acellular scaffold to promote new bone formation (26), serves as an additional nidus for bacterial colonization.
Coadministration of local diflunisal treatment and systemic vancomycin therapy during osteomyelitis.To determine the effects of diflunisal therapy on S. aureus-mediated bone destruction in the context of systemic antibiotic therapy, we analyzed cortical bone destruction of infected femurs treated with PUR+Veh, PUR+Dif+Veh, PUR+Vanc, and PUR+Dif+Vanc therapy (Table 1). Vancomycin was administered subcutaneously every 12 h at a dose of 10 mg/kg for 14 days postinfection. Diflunisal was delivered locally from a PUR foam with 20 mM diflunisal. At 14 days postinfection, infected femurs were removed and imaged by micro-computed tomography (μCT). Mice treated with PUR+Veh demonstrated significant cortical bone destruction (Fig. 4A and B). Consistent with previous studies (7), mice treated with PUR+Dif exhibited a significant (P < 0.01) decrease in cortical bone destruction. Yet both groups of mice treated with vancomycin demonstrated a significant decrease in cortical bone destruction regardless of whether local diflunisal therapy was given. Thus, combined therapy of diflunisal and vancomycin is capable of ameliorating the pathogenic bone destruction induced during staphylococcal osteomyelitis, but there was no additive osteoprotective benefit of local diflunisal delivery in the context of systemic antibiotic therapy.
Groups and doses of mice treated with vancomycin and diflunisal
Coadministration of local diflunisal treatment and systemic vancomycin therapy during osteomyelitis. (A) Micro-CT reconstructions of femurs treated with PUR+Veh, PUR+Dif+Veh, PUR+Vanc, or PUR+Dif+Vanc (see Table 1). n = 5 mice per group. One control group mouse suffered pathological fracture (second from left in the PUR+Veh group). (B) Quantification of cortical bone destruction. Error bar represents SD. **, P < 0.01; ***, P < 0.001 as determined by Student's t test.
DISCUSSION
Treatment of osteomyelitis and other musculoskeletal infections is hindered by the relatively poor penetration of antibiotics into the infectious focus (27–30). This is in part due to the ability of S. aureus to induce bone destruction, which leads to devascularized and necrotic segments of bone that can serve as a niche for chronic infection (5). Osteomyelitis therefore requires prolonged antibiotic treatment in conjunction with surgical debridement to increase the chance of cure. New adjunctive therapies that decrease pathogen-induced bone destruction might improve antibiotic activity by maintaining tissue integrity and preventing complications such as pathological fracture.
In this study, we evaluated the efficacy of combined systemic antibiotic therapy with local delivery of the quorum-quenching compound diflunisal, which we previously demonstrated to block S. aureus toxin-mediated cortical bone destruction (7, 8). We hypothesized that adjunctive diflunisal therapy would limit S. aureus-induced bone destruction without interfering with systemic antibiotic activity at the infectious focus. However, functional blockade of the Agr quorum sensing system increases biofilm formation in vitro, and it was therefore conceivable that quorum-quenching therapy would decrease antibiotic efficacy in vivo. Reconciling this hypothesis and potential outcome in a model of invasive staphylococcal disease is important given the exciting preclinical results with several quorum-quenching therapies (9–13). We discovered that systemic vancomycin therapy dose-dependently decreases bacterial burdens in our murine model of posttraumatic osteomyelitis. Importantly, diflunisal did not significantly alter vancomycin efficacy; however, combined treatment failed to improve antimicrobial activity or further reduce cortical bone destruction. Therefore, local diflunisal therapy is likely safe in the setting of systemic antimicrobial therapy, but there are no additive benefits for bacterial eradication or prevention of bone pathology under the experimental conditions deployed in this study. Given that diflunisal and vancomycin can be delivered concurrently in this preclinical model, investigation into possible synergistic efficacy of the two compounds is necessary. Moreover, measurement of diflunisal concentration in mouse serum following local administration is an important component of the preclinical testing that has yet to be performed. Further research will also focus on delayed antibiotic therapy to model a more antibiotic-recalcitrant infection. Similarly, experimentation with various doses of vancomycin, as well as other antibiotics, should be performed.
Compared to nonresorbable bone cements such as polymethylmethacrylate (PMMA), resorbable scaffolds offer the advantage of reduced bone grafting procedures and presumably minimize the presence of foreign material that could serve as a nidus for infection (25, 26, 31–33). PUR foams augmented with recombinant human bone morphogenetic protein (rhBMP-2) have been reported to support the regeneration of bone tissue (34, 35). While these foams degrade after 8 to 16 weeks in vivo (36, 37), our results suggest that even resorbable drug delivery platforms such as PUR foams can harbor significant numbers of bacteria during acute infection. Antibiotic-loaded PUR foams have been shown to decrease bacterial burdens (7, 26); however, tuning the release kinetics to deliver an effective dose for a sustained time period remains a major limitation. Dual delivery of diflunisal and antibiotic from a PUR foam has not yet been studied, yet the release of an antibiotic from the PUR surface may alleviate some of the bacterial colonization of the foreign body and affect the overall bone destruction outcome. As an alternative, bone-targeting antibiotics and nanoparticles have shown experimental promise and could be delivered systemically in patients requiring parenteral antibiotic therapy without the need for bone grafting (38, 39).
Further limitations of this study include the use of only one antibiotic, a single endpoint of 14 days, and use of a single infection model. Future studies should explore how quorum-quenching compounds might impact the activity of other anti-staphylococcal antibiotics to gain further understanding of potential synergistic combinations. Similarly, a focus on treatment timelines should be investigated to determine the effects of diflunisal therapy on antibiotic treatment duration. Diflunisal release from the PUR foams was slow (<1 wt%/day) after the initial burst release, and not all diflunisal loaded in the PUR released. It is unknown if more diflunisal released in vivo compared to the 20% of total loaded diflunisal released from a PUR foam in vitro. Although it is unclear if more diflunisal would ameliorate more bacteria-mediated bone destruction, alternative platforms that allow for repeat administrations of diflunisal should be investigated to understand the effect of higher doses on antibiotic activity at later time points. A limitation of the osteomyelitis model used in this study involved the high inoculum level required to induce infection. Although the exact bacterial load that initiates human osteomyelitis has yet to be definitively determined and may vary significantly depending on the mechanism of disease, it is likely that many patients experience a much lower initial inoculum. Furthermore, other murine musculoskeletal infection models have shown qualitative differences in the immune responses to S. aureus in bone based on the initial infectious burden (40). Another limitation of this study was the relatively small sample sizes used in the in vivo experiments, which may have limited our ability to detect more subtle clinical outcomes. A final limitation of this study is that we did not test the effect of diflunisal therapy on implant-associated osteomyelitis, where biofilm formation might play a greater role in treatment recalcitrance. Nevertheless, although the delivery of diflunisal and vancomycin can be improved, our work suggests that combined diflunisal and vancomycin treatment warrants further study as a potential adjunctive antivirulence therapy for osteomyelitis.
MATERIALS AND METHODS
Bacterial strains, reagents, and growth conditions.An erythromycin-sensitive derivative of the methicillin-resistant S. aureus (MRSA) USA300 lineage strain LAC was used for all experiments, as it represents the most commonly isolated clonal complex causing musculoskeletal infection in the United States (41, 42). Overnight cultures were grown in tryptic soy broth (TSB) at 37°C and 180 rpm shaking. Optical density at 600 nm (OD600) was measured to determine bacterial growth prior to infection. Diflunisal was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in 100% DMSO at a final concentration of 10 mg/ml. Lysine triisocyanate (LTI) was purchased from Jinan Haohua Industry Co., Ltd. (Jinan, China) and refluxed with a dispersion of activated carbon (Fisher Scientific) in t-butyl methyl ether (TBME; Acros Organics) at 60°C for 22 h to remove high-molecular-weight impurities (43). For polyester triol synthesis, ε-caprolactone and stannous octoate were purchased from Sigma-Aldrich, and D,L-lactide and glycolide were purchased from Polysciences (Warrington, PA). Triethylene diamine (TEDA) catalyst was received from Goldschmidt (Tegoamin 33; Hopewell, VA). All other reagents, including calcium stearate and turkey red oil, were purchased from Sigma-Aldrich. Vancomycin-HCl was purchased from GoldBio (St. Louis, MO).
Synthesis of PUR foams for local drug delivery.PUR foams were synthesized as previously reported (7). Briefly, a polyester triol (polyol) was synthesized containing 70% ε-caprolactone, 20% glycolide, and 10% lactide (Mn = 900 g mol−1). The polyester triol and LTI were reacted to form a settable PUR foam. Polymer components were mixed in a 5-ml plastic container for 1.5 min using a Hauschild SpeedMixer DAC 150 FVZ-K vortex mixer (Flacktek) and left overnight to cure. Blank foams and foams loaded with 10 mM (1.3 wt%) or 20 mM (2.6 wt%) diflunisal (based on foam volume) were tested. Foams were cut into prisms (8 mm by 4.5 mm by 2 mm) for in vivo testing and sterilized by γ-irradiation (25 kGy). Foams were imaged by SEM to characterize pore size and porosity using ImageJ.
In vitro release kinetics of diflunisal PUR foams.Specimens for in vitro testing were cut to match the size of those tested in vivo. Foams containing 20 mM diflunisal were submerged in 8 ml of PBS in a glass vial and incubated at 37°C with slight agitation. Four milliliters of leachate were removed on days 1, 2, 3, 5, 11, and 14 and replaced with 4 ml of fresh PBS. We previously evaluated the release of diflunisal from PUR by high-pressure liquid chromatography (HPLC) (7). We previously analyzed the cumulative diflunisal release relative to the total amount released from the foam by day 14. In the current study, we instead evaluated diflunisal release by the amount of diflunisal released as a fraction of the theoretical mass loaded in each foam. Diflunisal concentration was quantified using a UV-vis plate reader with a fluorescence reading at 420 nm and excitation of 310 nm (44). The release of diflunisal was characterized by the cumulative release percentage of diflunisal compared to the total loading in each foam sample.
Bacterial viability assay.Bacterial viability following exposure to various concentrations of vancomycin and diflunisal was assessed in nontreated polystyrene 24-well plates. S. aureus was cultured overnight and diluted to an initial inoculum of 105 CFU/cm2. Diflunisal and vancomycin were solubilized by DMSO and diluted in TSB. Bacteria were exposed to various concentrations of diflunisal and/or vancomycin for 24 h at 37°C while agitated at 80 rpm. Adherent bacteria were then washed with sterile PBS and separated from the plate surface using a sonicator bath. Sonicated samples were serially diluted and plated on TSA plates for CFU enumeration per surface area of well plate.
Preparation of concentrated supernatants.Concentrated supernatants were prepared as previously reported (7). Briefly, 3 colonies were inoculated into triplicate 50-ml cultures in capped 250-ml Erlenmeyer flasks and grown for 15 h in RPMI medium and 1% Casamino Acids together with PUR foams containing 0, 10, 20 mM diflunisal, 10 μg/ml diflunisal, or 10 μg/ml diflunisal with 0.1 μg/ml vancomycin. DMSO was used as a vehicle control for solubilized diflunisal and vancomycin delivery. Bacteria were grown at 37°C for 15 h and 180 rpm, after which a sample was taken for CFU enumeration and supernatants were separated by centrifugation. Supernatants were sterilized by passage through a 0.22-μm filter and then concentrated to a final volume of ∼1.5 ml using an Amicon Ultra 3-kDa nominal molecular weight column. Supernatants were again filter sterilized and frozen at −80°C.
Osteoblast cytotoxicity assay.Osteoblast cytotoxicity was assessed using MC3T3-E1 murine osteoblast cells as previously reported (7). Briefly, cells were seeded in 96-well tissue culture-treated plates and grown at 37°C and 5% CO2. Cells were intoxicated with prepared supernatants 12 to 24 h after seeding and then incubated for 22 h, at which time cell viability was determined using CellTiter Aqueous One solution (Promega, Madison, WI) according to the manufacturer’s instructions.
Murine model of osteomyelitis and systemic vancomycin treatment.This study was approved by the Institutional Animal Care and Use Committee of Vanderbilt University Medical Center and conducted in compliance with Animal Welfare Regulations and the principles of the Guide for the Care and Use of Laboratory Animals. Osteomyelitis was induced in 7- to 8-week-old female C57BL/6J mice as previously described (5). An inoculum of ∼1 × 106 CFU in 2 μl PBS was delivered into murine femurs. To determine a systemic vancomycin dose that does not result in complete eradication of infection, mice (n = 5) were subcutaneously injected with 50 μl of 0, 10, 20, or 30 mg/kg vancomycin in sterile PBS every 12 h. Mice were euthanized 7 days postinfection, and the infected femur was removed and processed for CFU enumeration. Femurs were homogenized in a Bullet Blender (Next Advance, Averill, NY) using the Navy Bead Lysis kit and plated at limiting dilution on tryptic soy agar (TSA). Results are reported as log10 CFU/femur.
Combined systemic vancomycin and local diflunisal treatment in a murine model of osteomyelitis.Osteomyelitis was induced in 7- to 8-week-old female C57BL/6J mice as described above (5). We injected 10 mg/kg of vancomycin subcutaneously (SQ) every 12 h. Sterile PBS was injected as a vehicle control. PUR foams (8 mm by 4.5 mm by 2 mm) containing 20 mM diflunisal were fabricated, sterilized, and wrapped around the femur at the inoculation site and sutured into place. Empty PUR foams served as a mock control. Groups are identified in Table 1. Mice that experienced >20% weight loss following infection were euthanized. For evaluation of bacterial burdens, mice were euthanized at 7 days postinfection, and the infected femur was removed and processed for CFU enumeration as above. To evaluate bacterial dissemination, livers, kidneys, and foams were also removed and processed for CFU enumeration. Residual PUR foam was separated from soft tissues and sonicated in an ultrasonic bath for 5 min prior to CFU enumeration. To evaluate cortical bone destruction, additional groups of mice were euthanized at day 14 postinfection, and the infected femurs were analyzed by μCT as described previously (5). Briefly, axial images of each femur were captured with 5.0-μm voxels at 70 kV, 200 μA, 2,000 projections per rotation, and an integration time of 350 ms in a 10.24-mm field of view. Each imaging scan comprised 1,635 slices (8.125 mm) of the length of the femur, centered on the inoculation site as visualized in the scout-view radiographs. Volume of interest (VOI) was limited to the original cortical bone, and any destruction was selected by drawing inclusive contours on the periosteal surface, excluding contours on the endosteal surface.
Statistical evaluation.Differences in CFU counts and cortical bone destruction were analyzed by Student's t test. A P value of ≤ 0.05 was considered significant.
FOOTNOTES
- Received 27 January 2020.
- Returned for modification 1 March 2020.
- Accepted 22 April 2020.
- Accepted manuscript posted online 27 April 2020.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
