TY - JOUR
AU - PhD, Warren O Haggard,
AB - Abstract 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. INTRODUCTION Bone fixation devices may be required to ameliorate traumatic injuries and some procedures to the musculoskeletal system. According to estimates, open fracture and extremity trauma are approximated to be six million or more each year in the USA.1 U.S. military personnel have combat-related infection rates ranging from 5.5% to 49%.2 Infection rates of 5% are seen when fixation devices are implemented in musculoskeletal injuries,3 increasing socioeconomic costs,4 risk of antibiotic resistance, and rates of morbidity and mortality in U.S. Military Service members.5 More than 65% of infections being treated in the developed world are caused by biofilm-forming bacteria.6 Injuries that result from combat-related blasts possess a high probability of infection due to the complexity of the wounds and the existence of environmental contaminants, with some studies showing biofilm forming on similar injuries within a matter of hours.7 Injuries received on the battlefield are further convoluted when polymicrobial infection containing Gram-negative and Gram-positive bacteria are involved.7,8Escherichia coli and Pseudomonas aeruginosa were two of the more frequent bacterial strains reported during recent military operations, with verified presence in 14% of wounds.8 Incorporating biofilm-targeting antibiotics into engineered inexpensive biodegradable devices could be advantageous in preventing infection, especially where biofilm-prone fracture fixation devices are used.9–12 Infection prevention after an injury is a principal strategy in managing combat-related trauma, with administration of systemic antibiotics within 3 h of injury to reduce the chance of wound infection and bacterial colonization being the primary care option utilized.7,13 Local delivery systems for antibiotics, such as antibiotic-loaded poly(methyl methacrylate) beads (PMMA) and calcium sulfate, are growing in acceptance as an adjunctive approach to systemic delivery.14–16 Local delivery systems may be advantageous over systemic administration in that they deliver higher concentrations of antibiotic directly to the affected tissue14 and minimize risk of toxicity to organs.15,17–19 PMMA beads are non-degradable and require an additional surgery to remove the implanted material for complete wound healing. Additionally, once PMMA beads have released their antibiotic load, they provide surfaces for potential biofilm formation.20 Rapid resorption of calcium sulfate pellets results in calcium-rich fluid that incites an inflammatory response and wound drainage.21 Local delivery devices that allow for practicing clinicians to load with broad spectrum or multiple antibiotics at the time of intervention may reduce polymicrobial contamination. Benefits of local antibiotic delivery systems could include an extended release profile, biocompatibility, biodegradability with biocompatible degradation products, and personalized antimicrobial selection depending on the individual and what is being targeted.22 Chitosan, an adaptable natural biopolymer, has been used to fabricate a myriad of drug delivery systems,22–24 including lyophilized sponges.25 Commercially available chitosan wound dressings in the form of a sponge were approved by the U.S. Food and Drug Administration (FDA) for wound management, possessing the ability to be hydrated with solutions for applications involving varying degrees of musculoskeletal wounds. Practicing physicians, at their clinical discretion, may choose to include antibiotics in the hydrating solution in their treatment solution of contaminated or infected traumatic injuries.26,27 FDA clearance of antibiotic introduction into the hydrating solution has not been specifically obtained. Chitosan sponges have shown excellent biocompatibility, degradability, and may be loaded with many antibiotics by passive absorption and diffusion.10,22,25,27,28 Sponge fabrication allows for varying thicknesses and pore sizes using a lyophilization process, allowing the release of antibiotic solutions to be tailorable,25,29 with geometry that can be customized for treatment of complex wounds. Specific biofilm-targeting antibiotics may be particularly useful for infection prevention when delivered from a local delivery device. Rifampin (Rif) has emerged as a major player in antibiotic combination therapy to combat biofilm among other infections. Rifampin specifically inhibits synthesis of bacterial proteins,30,31 with activity against Gram-positive bacteria. It has been shown that Rif diffuses well into biofilm where it acts to kill biofilm-associated bacteria.31 Rifampin-resistant mutants may be selected, so guidelines suggest that rifampin should be used in conjunction with other antibiotics.31 In this study, ciprofloxacin (Cipro), a 2nd generation fluoroquinolone, was selected for proven efficacy against Gram-negative and some Gram-positive bacterial pathogens.32–40 This study seeks to investigate the effects of combining Cipro and Rif for local delivery from chitosan sponges for infection prevention, as a result of the incidence of polymicrobial biofilm and antibiotic resistance. MATERIALS AND METHODS In vitro and in vivo tests were used to evaluate the efficacy of combined therapy of ciprofloxacin and rifampin against pathogens commonly associated with infection resulting from complex musculoskeletal wounds and/or implants. For each in vitro test that required a specific sample size to obtain the desired power separate, independent, randomly chosen, and mutually exclusive sponges were used. Chitosan Sponges Chitosan sponges containing 2.0% chitosan or 1.5% chitosan and 0.5% poly(ethylene glycol) (PEG) were fabricated and used in all in vitro studies alongside BioSponge except for the in vivo implant-associated biofilm model. BioSponge was the only chitosan sponge used for the in vivo animal model. Fabricated chitosan sponges, for all in vitro studies, were made by dissolving either 2.0% chitosan (w/v) or 0.5% PEG and 1.5% chitosan (w/v) in 1% acidic (v/v) solutions.25,41 The solutions were placed in template containers with the desired shape and thickness and frozen at −20°C. Once frozen, the sponges were lyophilized, neutralized, frozen again, re-lyophilized and then sterilized (Sterigenics, West Memphis, AR, USA) using low-dose gamma radiation (25–40 kGy). Chitopharm S chitosan was obtained from Chitinor AS (Tromsoe, Normway) having an 82 ± 2 DDA, 251 ± 17 kDa weight-average molecular weight (MW) and 2.013 ± 0.145 polydispersity index. PEG was purchased from Sigma Aldrich (St. Louis, MO, USA) having a 6 kDa MW. Sentrex BioSponge (Bionova Medical, Germantown, TN, USA) was included in the study due to their clinical use and commercial availability. Antibiotic Dissolution Targeted applications of the studies incorporated to test the efficacy of the Cipro/Rif combination required that the antibiotics be in solution. The dissolution of rifampin proved to be the most challenging aspect. Preliminary elution studies failed to address the solubility issue and the results (not shown) were comparable to prior studies.12,41 However, it was later shown that rifampin did not have an acceptable dissolution for our targeted applications. Rifampin and its solubility was addressed by using 0.1 N hydrochloric acid at 37°C with a final pH of 6.02. Scanning electron microscopy (SEM) images were taken to ensure that the solution pH value did not affect the structure or integrity of the chitosan sponges (Figs 1–3). FIGURE 1. View largeDownload slide SEM images of the BioSponge pre-hydration (top) and post-hydration (bottom). The sponge was hydrated in antibiotic solution for 10 min, which may be comparable for times in a clinical environment. As shown, the pH of the hydrating solution was not adversely breaking down the structure. FIGURE 1. View largeDownload slide SEM images of the BioSponge pre-hydration (top) and post-hydration (bottom). The sponge was hydrated in antibiotic solution for 10 min, which may be comparable for times in a clinical environment. As shown, the pH of the hydrating solution was not adversely breaking down the structure. FIGURE 2. View largeDownload slide SEM images of the 0.5% PEG/1.5% chitosan sponge pre-hydration (top) and post-hydration (bottom). The sponge was hydrated in antibiotic solution for 10 min, which may be comparable for times in a clinical environment. As shown, the pH of the hydrating solution was not adversely breaking down the structure. FIGURE 2. View largeDownload slide SEM images of the 0.5% PEG/1.5% chitosan sponge pre-hydration (top) and post-hydration (bottom). The sponge was hydrated in antibiotic solution for 10 min, which may be comparable for times in a clinical environment. As shown, the pH of the hydrating solution was not adversely breaking down the structure. FIGURE 3. View largeDownload slide SEM images of the 2.0% chitosan sponge pre-hydration (top) and post-hydration (bottom). The sponge was hydrated in antibiotic solution for 10 min, which may be comparable for times in a clinical environment. As shown, the pH of the hydrating solution was not adversely breaking down the structure. FIGURE 3. View largeDownload slide SEM images of the 2.0% chitosan sponge pre-hydration (top) and post-hydration (bottom). The sponge was hydrated in antibiotic solution for 10 min, which may be comparable for times in a clinical environment. As shown, the pH of the hydrating solution was not adversely breaking down the structure. In Vitro Elution BioSponge, 2.0% chitosan, and 0.5% PEG/1.5% chitosan sponges were sectioned and weighed to nominal masses of 25 mg. The sponges (n = 5/group) were loaded with 5 mg mL−1 of ciprofloxacin (Acros Organics, Geel, Belgium) and 5 mg mL−1 of rifampin (Fisher Scientific, Fair Lawn, NJ, USA), the solvent was sterile 1X phosphate buffered saline (PBS). Each sponge was immersed in abundant ciprofloxacin and rifampin antibiotic solution (~10 mL) to allow hydration saturation. Sponge pre- and post-hydration masses were recorded in order to calculate approximate antibiotic solution volume absorbed into the samples. The sponge sections were gently placed within 125 mL NALGENE containers with no impedance to natural structure, which allowed for adequate coverage by solution. Sterile 1X PBS (30 mL) was used to submerge the antibiotic-hydrated sectioned sponges. The submerged sponges were placed in an incubator (LabDoctor Mini Incubated Shaker, MidSci, ST. Louis, MO, USA) at 37°C under constant motion (30 rpm). Samples of 15 mL each were taken at 1, 2, 3, 4, 5, 6, and 7 d. When the samples were taken, the PBS solution was refreshed. Refreshed samples were returned to incubator until their time period was reached, at which point the process repeated until all time points were elapsed. All sample collection and PBS exchange was conducted in an aseptic environment. Once samples were collected they were stored for eluate concentration analysis. Eluate sample storage was at −20°C to prevent antibiotic degradation and maintain antibiotic activity. Eluate Concentration Analysis High-pressure liquid chromatography (HPLC), (UltiMate 3000, Thermo Scientific, West Palm Beach, FL, USA) was the analytical technique used to quantify the concentration of antibiotics in the sponge eluates. HPLC analysis for both antibiotics was modeled after a method used by Liu et al.42 Chromatographic separation was achieved by using a mixture of methanol-acetonitrile-dipotassium phosphate (55 mM)-phosphoric acid (1.0 M) (28:30:38:4, v/v) as the primary mobile phase (MP1), in addition to a buffer solution consisting of 55 mM K2HPO4 and 1.0 M H3PO4 (MP2). Ciprofloxacin was read at λ = 280 bandwidth of 4 with a retention time of 2.38 min. Rifampin was read at λ = 333 bandwidth of 4 with a retention time of 11.21 min. A gradient method was utilized with at a flow rate of 1.5 mLmin−1. Detection of ciprofloxacin occurred with MP2 at 60% and MP1 at 40% the gradient was then ramped up at 3 min to 100% MP1 for the detection of rifampin. The concentrations of the antibiotics were measured to determine their elution profile over time.42–54 In Vitro Antibiotic Activity Pseudomonas aeruginosa (ATCC 27317) was grown overnight at 37°C in trypticase soy broth (TSB). The overnight growth was diluted 1:50 in TSB and 100 μL of this was added to plates of TSB agar and spread to make a lawn of bacteria. Once the lawn of bacteria was complete 6 mm blank disks (Beckton, Dickinson cat # 231039) were placed on the agar plates. Subsequent to the placement of the disks was the addition of 20 μL of the sponge eluate (n = 5/group) to the disks. The plates were incubated for 24 h at 37°C, pictures were taken and the diameter of the zone of inhibition (ZOI) was recorded. Each plate contained five replicate eluate samples for each group and time point. S. aureus (Cowan I ATCC 12598) was grown and tested exactly as P. aeruginosa but with a dilution of 1:10. In Vitro Synergy Assay Checkerboard synergy testing was performed using 96-well microtiter plates. Ciprofloxacin and rifampin were dissolved in PBS and diluted in tryptic soy broth (TSB) in seven 2-fold dilutions from 4 μg mL−1 for ciprofloxacin plated against P. aeruginosa (ATCC 27317) and from 2 μg mL−1 plated against S. aureus (UAMS-1). UAMS-1 was selected during this step because this clinical isolate has been shown to form biofilm in microtiter assays.55 Rifampin concentrations were from 256 μg mL−1 against S. aureus (UAMS-1) and from 0.06 μg mL−1 against P. aeruginosa (ATCC 27317). Each solution was pipetted into triplicate wells of the 96-well plate. Positive and negative controls for this experiment were inoculating TSB, without antibiotics, with and without bacteria, respectively. Each well was inoculated with S. aureus (UAMS-1) or P. aeruginosa (ATCC 27317) for a final concentration of approximately 1 × 104 CFUs per well. Plates were incubated for 24 hours at 37°C. Planktonic bacteria were removed from the wells through aspiration of the liquid without disturbing the biofilm followed by gently washing with PBS three times. Heat fixation of the biofilm bacteria was achieved by heating the microtiter plate at 60°C for 1 h. Once the biofilm was fixed, it was stained using 100 μL of crystal violet solution. Crystal violet not absorbed by the biofilm was removed by gently rinsing with water. At that time, a de-staining solution composed of 7.5% acetic acid, 10% methanol, and water was used to dissolve the absorbed crystal violet. To conclude, absorbance measurements were obtained at λ = 540 nm using a plate reader spectrophotometer (Biotek ELx800, Winooski VT, USA). The minimum biofilm inhibitory concentration (MBIC) is the concentration of antibiotic required to inhibit the formation of biofilm. Relationships between antibiotics were quantified using the fractional inhibitory concentration index (FICI).56,57 FICI was determined for each antibiotic as follows, the MBIC for the antibiotic in combination was divided by the MBIC of each antibiotic alone. The FICI for each antibiotic was summed to acquire a final FICI value. FICI values <1 were considered synergistic, ≥1 and <2 were additive, = 2 were indifferent, and >2 were antagonistic. In Vivo Implant-Associated Biofilm Model All methods were approved and monitored for compliance by the animal use committee at UAMS and by the Animal Care Use and Review Office (ACURO) at the USAMRMC. A pilot murine model with orthopedic implant-associated infection58 was adapted to use a polymicrobial mixture of S. aureus (UAMS-1)59 and E. coli (ATCC 25922).60 The combination of these two bacteria was chosen due to the reported use of this Gram-negative strain in previously published works.60 In the infected mouse pin model (UAMS – IACUC 3579), sterilized chitosan sponges were hydrated with antibiotic solutions. The sponges were hydrated with 5 mL of an antibiotic solution combination of ciprofloxacin and rifampin (10 mg mL−1 each). C57BL/6 mice, 8–12 wk old, 16–18 grams in weight were anesthetized with Isoflurane and Avertin (400–600 μg g−1) and the adequacy of the anesthesia was confirmed by the toe pinch reflex and the reaction to light shined into the mice eyes. The left leg was cleaned with povidine iodine and rinsed with 70% ethanol. An incision was made at the knee, a hole was drilled into the left distal femur with a 26-gauge syringe needle followed by a 23-gauge syringe needle. A sterile 1 cm × 600 μm diameter stainless steel Kirschner wire was inoculated with approximately 104 colony forming units (CFUs) of S. aureus (UAMS-1) and 102 CFUs of E. coli (ATCC 25922) and inserted into the femur. Placement of the pin within the femur can be seen in Figure 4. FIGURE 4. View largeDownload slide Pictured is an X-ray that shows the placement of the pin within the left femur. The arrow points to the point of insertion. FIGURE 4. View largeDownload slide Pictured is an X-ray that shows the placement of the pin within the left femur. The arrow points to the point of insertion. Chitosan sponges loaded with saline (negative control) or the combination of ciprofloxacin and rifampin at 10 mg mL−1 each were implanted adjacent to the contaminated implant (n = 12 per group). After surgical site closure, the mice were returned to their cages, monitored daily, and any that appeared moribund would be euthanized using CO2. None of the animals displayed any dilapidated attributes during the duration of the study; the design and goal of the study was survival until Day 7.60 Animals were euthanized 7 d after the treatment, the wire implant and any associated femur tissue were removed for determination of viable bacterial CFUs remaining on/in each. The soft tissue was dissected from the bone and the femur was cut into small pieces, homogenized, and placed in a sterile saline. Wire implants were sonicated and vortexed in sterile PBS. After overnight incubation at 37°C, the homogenates were diluted, plated on agar plates, and the viable microbial colonies were counted along with the colonies remaining on the pin and bone. Bacterial clearance was defined as an apparent bacterial CFU count of zero. Statistical Analysis For in vitro analyses of eluate concentration and activity, in order to detect a mean difference of 6 μg mL−1 with an estimated standard deviation of 2.5 μg mL−1 for 0.80 power at α = 0.05 a sample size of five was required per group. The analysis was based on elution data obtained from previous studies conducted within the lab. For in vivo studies, an appropriate sample size determination was used to determine that 6 animals in each group were required to have 80% power to detect a difference in proportions completely cleared of bacteria of 90% at α = 0.05. The study was repeated for validity to bring the total sample size to 12/group and allowed for detection of differences in rates of contamination of 60% or more at the 5% significance level. Statistical analysis of results was performed using Sigma Plot (Systat Software, Inc, San Jose, CA, USA). One way Kruskal Wallis Analysis of Variance (ANOVA) was used to determine differences between CFUs retrieved from implants, with p values of <0.05 being considered statistically significant. Dunn’s post hoc analysis was used to compare groups pairwise and determine p values. Fisher exact tests of contingency tables were used to compare the rate of clearance for implants in antibiotic-loaded groups to controls, with Bonferroni post hoc correction in cases of multiple comparisons. RESULTS In Vitro Elution Results from the elution study showed that rifampin elutes from its delivery device within 3 d (Fig. 5). Ciprofloxacin’s elution profile while still displaying a burst effect was sustained throughout the duration of the study (Fig. 6). FIGURE 5. View largeDownload slide The elution profile of rifampin when released from chitosan sponges. The concentration was determined by chromatographic separation using a gradient based HPLC method. Rifampin releases fast and strong within the first 3 d. At this point efforts were not made to tailor the release profile since there was another broad-spectrum antibiotic being used in conjunction that was able to sustain a release profile throughout the duration. Data are represented as mean ± standard deviation. FIGURE 5. View largeDownload slide The elution profile of rifampin when released from chitosan sponges. The concentration was determined by chromatographic separation using a gradient based HPLC method. Rifampin releases fast and strong within the first 3 d. At this point efforts were not made to tailor the release profile since there was another broad-spectrum antibiotic being used in conjunction that was able to sustain a release profile throughout the duration. Data are represented as mean ± standard deviation. FIGURE 6. View largeDownload slide The elution profile of ciprofloxacin while experiencing an initial burst was sustained at levels above its reported minimum inhibitory concentration for the duration of the study. The concentration was determined with chromatographic separation using a gradient HPLC method. Ciprofloxacin’s ability to target both Gram-negative and certain Gram-positive bacteria was a compliment to rifampin, which released completely within 3 d. Data are represented as mean ± standard deviation. FIGURE 6. View largeDownload slide The elution profile of ciprofloxacin while experiencing an initial burst was sustained at levels above its reported minimum inhibitory concentration for the duration of the study. The concentration was determined with chromatographic separation using a gradient HPLC method. Ciprofloxacin’s ability to target both Gram-negative and certain Gram-positive bacteria was a compliment to rifampin, which released completely within 3 d. Data are represented as mean ± standard deviation. In Vitro Antibiotic Activity Eluates from chitosan based local delivery devices were able to inhibit P. aeruginosa (ATCC 27317) (Fig. 7) and S. aureus (ATCC 12598) (Fig. 8) through the duration of the study 7 d when solubility was addressed. Chitosan sponge eluates from POC elution were able to inhibit, on average 4 d, P. aeruginosa (ATCC 27317) (Fig. 7) and S. aureus (ATCC 12598) (Fig. 8). FIGURE 7. View largeDownload slide ZOI results of sponge eluates tested against P. aeruginosa (ATCC 27317). On the left of each sponge group were the results before the solubility issue was addressed. On average, 3 d of inhibition was seen in each group. On the right, it demonstrates that inhibition was achieved through the duration of the study for each group. The plates on the right were achieved as the solubility of rifampin was actively addressed. FIGURE 7. View largeDownload slide ZOI results of sponge eluates tested against P. aeruginosa (ATCC 27317). On the left of each sponge group were the results before the solubility issue was addressed. On average, 3 d of inhibition was seen in each group. On the right, it demonstrates that inhibition was achieved through the duration of the study for each group. The plates on the right were achieved as the solubility of rifampin was actively addressed. FIGURE 8. View largeDownload slide ZOI results of sponge eluates tested against S. aureus (Cowan I ATCC 12598). On the left of each sponge group were the results before the solubility issue was addressed. On average, 4 d of inhibition was seen in each group. On the right, it demonstrates that inhibition was achieved through the duration of the study for each group. The plates on the right were achieved as the solubility of rifampin was actively addressed. FIGURE 8. View largeDownload slide ZOI results of sponge eluates tested against S. aureus (Cowan I ATCC 12598). On the left of each sponge group were the results before the solubility issue was addressed. On average, 4 d of inhibition was seen in each group. On the right, it demonstrates that inhibition was achieved through the duration of the study for each group. The plates on the right were achieved as the solubility of rifampin was actively addressed. In Vitro Synergy Assay Results from the synergy assay showed an additive effect between ciprofloxacin and rifampin against P. aeruginosa (ATCC 27317) with a FICI value of 1 (Table I) and no discernible effect against S. aureus (UAMS-1) (Table I). Table I. FICI Values and Interpretation for Ciprofloxacin and Rifampin Against S. aureus and P. aeruginosa Bacteria  Ciprofloxacin MBIC (Alone, μg mL−1)  Ciprofloxacin MBIC (Combo, μg mL−1)  Rifampin MBIC (Alone, μg mL−1)  Rifampin MBIC (Combo, μg mL−1)  FICI  Combined Effect  S. aureus (UAMS-1)  0.0625  0.0313  0.0019   0.0009   N/A  Not determined  P. aeruginosa (ATCC 27317)  0.125  0.0625  128   64   1  Additive  Bacteria  Ciprofloxacin MBIC (Alone, μg mL−1)  Ciprofloxacin MBIC (Combo, μg mL−1)  Rifampin MBIC (Alone, μg mL−1)  Rifampin MBIC (Combo, μg mL−1)  FICI  Combined Effect  S. aureus (UAMS-1)  0.0625  0.0313  0.0019   0.0009   N/A  Not determined  P. aeruginosa (ATCC 27317)  0.125  0.0625  128   64   1  Additive  Table I. FICI Values and Interpretation for Ciprofloxacin and Rifampin Against S. aureus and P. aeruginosa Bacteria  Ciprofloxacin MBIC (Alone, μg mL−1)  Ciprofloxacin MBIC (Combo, μg mL−1)  Rifampin MBIC (Alone, μg mL−1)  Rifampin MBIC (Combo, μg mL−1)  FICI  Combined Effect  S. aureus (UAMS-1)  0.0625  0.0313  0.0019   0.0009   N/A  Not determined  P. aeruginosa (ATCC 27317)  0.125  0.0625  128   64   1  Additive  Bacteria  Ciprofloxacin MBIC (Alone, μg mL−1)  Ciprofloxacin MBIC (Combo, μg mL−1)  Rifampin MBIC (Alone, μg mL−1)  Rifampin MBIC (Combo, μg mL−1)  FICI  Combined Effect  S. aureus (UAMS-1)  0.0625  0.0313  0.0019   0.0009   N/A  Not determined  P. aeruginosa (ATCC 27317)  0.125  0.0625  128   64   1  Additive  In Vivo Implant-Associated Biofilm Model The combination of ciprofloxacin and rifampin was able to completely clear both the pin (Fig. 9) and the bone (Fig. 10) of S. aureus CFUs at 7 d. S. aureus clearance can be seen with SEM images (Fig. 11). While some E. coli colonies were retrieved from the bone tissue in negative saline controls without loaded antibiotics (Fig. 12), there were no viable colonies after 7 d on the implant (Fig. 13). FIGURE 9. View largeDownload slide The combination of ciprofloxacin and rifampin was able to completely clear the pin of viable CFUs. The concentration used during the murine model was 10 μg mL−1 for each antibiotic. The PBS pin did not have any antibiotic loading. FIGURE 9. View largeDownload slide The combination of ciprofloxacin and rifampin was able to completely clear the pin of viable CFUs. The concentration used during the murine model was 10 μg mL−1 for each antibiotic. The PBS pin did not have any antibiotic loading. FIGURE 10. View largeDownload slide The combination of ciprofloxacin and rifampin was able to completely clear the bone of viable CFUs. The concentration used during the murine model was 10 μg mL−1 for each antibiotic. The PBS pin did not have any antibiotic loading. FIGURE 10. View largeDownload slide The combination of ciprofloxacin and rifampin was able to completely clear the bone of viable CFUs. The concentration used during the murine model was 10 μg mL−1 for each antibiotic. The PBS pin did not have any antibiotic loading. FIGURE 11. View largeDownload slide SEM image taken of the pin for a PBS pin with no antibiotic loading (top) with a discernible presence of S. aureus. Ciprofloxacin and rifampin treated pin (bottom) SEM image reveals no structures with the appearance of S. aureus. FIGURE 11. View largeDownload slide SEM image taken of the pin for a PBS pin with no antibiotic loading (top) with a discernible presence of S. aureus. Ciprofloxacin and rifampin treated pin (bottom) SEM image reveals no structures with the appearance of S. aureus. FIGURE 12. View largeDownload slide Ciprofloxacin and rifampin were able to clear the bone of E. coli CFUs in comparison with PBS containing no antibiotics. It is noted that this particular strain of E. coli did not perform as desired. The concentration of both antibiotics was 10 μg mL−1. FIGURE 12. View largeDownload slide Ciprofloxacin and rifampin were able to clear the bone of E. coli CFUs in comparison with PBS containing no antibiotics. It is noted that this particular strain of E. coli did not perform as desired. The concentration of both antibiotics was 10 μg mL−1. FIGURE 13. View largeDownload slide There were no viable E. coli CFUs on the pin for either group. Since the PBS groups contained neither antibiotic nor any other deterrent for inhibition of growth this demonstrates a limitation in the study. The strain of E. coli chosen was not robust and did not readily form biofilm. Further investigations are needed in order to validate if the combination is effective in a polymicrobial model consisting of both Gram-negative and Gram-positive bacteria. The concentration of both antibiotics was 10 μg mL−1. FIGURE 13. View largeDownload slide There were no viable E. coli CFUs on the pin for either group. Since the PBS groups contained neither antibiotic nor any other deterrent for inhibition of growth this demonstrates a limitation in the study. The strain of E. coli chosen was not robust and did not readily form biofilm. Further investigations are needed in order to validate if the combination is effective in a polymicrobial model consisting of both Gram-negative and Gram-positive bacteria. The concentration of both antibiotics was 10 μg mL−1. DISCUSSION The primary purpose of this study was to ascertain if local drug delivery of Cipro and Rif from chitosan sponges could assist with inhibiting biofilm formation. During this study, a combination of the antibiotics was actively released from chitosan sponges at levels that were shown to be inhibitory. This exhibits that this combination may be introduced into a local delivery system in a clinical setting and release active concentrations for infection inhibition. Study results suggest a potential additive relationship against Gram-negative P. aeruginosa for ciprofloxacin and rifampin. Gram-negative bacteria, specifically P. aeruginosa, have a history of being persistent.61,62 Treatment options consisting of antibiotics with different mechanisms of action that are additive may eliminate the persister cells in potential clinical applications. No discernible effects, synergistic, additive, antagonistic, or indifferent, were obtained against the Gram-positive S. aureus due to the combination completely inhibiting the in vitro adherence after some growth. While the ability to inhibit biofilm was not determined by the in vitro synergy assay it was shown that this combination can inhibit growth of S. aureus. The efficacy of this treatment method was further investigated using a known model of implant infection. Results revealed complete clearance of Gram-positive S. aureus when dual-loaded chitosan sponges were introduced. Rifampin has been tested with a number of other antibiotics for potential synergistic effects against Acinetobacter baumannii,63Klebsiella pneumoniae,64 and S. aureus65 among others. Ciprofloxacin has been investigated in conjunction with several antibiotics against P. aeruginosa,66Enterbacteriaceae and D. streptococcal67 to name a few. Very few studies have reported the effects of the combination of ciprofloxacin and rifampin against the strains of bacteria tested during this study. In a study by Zimmerli et al,68 a cure rate of 100% was achieved for those in the ciprofloxacin-rifampin group in patients with culture-proven Staphylococcal infection associated with stable orthopedic implants. Ciprofloxacin and rifampin were the most effective treatment regimen in reduction of MRSA in bone in rats with experimental osteomyelitis.69 The groups tested were vancomycin, vancomycin-rifampin, ciprofloxacin, ciprofloxacin-rifampin, rifampin, and a control group.69 Widmer et al70 concluded that combination therapy with rifampin and a quinolone should be considered for patients with orthopedic implant-related infections if the implant cannot be removed. The Widmer study centered around patients with infections resulting from staphylococci or streptococci.70 The additive effect against P. aeruginosa shows promise for this combination. Complete inhibition of S. aureus was validated with results from most phases of the study with culmination coming from the implant-associated biofilm model. Biofilm synergy assay results for S. aureus were not able to be determined as the concentrations of the antibiotics completely inhibited growth. Future investigations are planned with lower concentrations of both antibiotics. Delivery of the dual antibiotic combination amikacin and vancomycin has been shown to completely clear P. aeruginosa and S. aureus on implants.10,71,72 Broad-spectrum antibiotics, such as the aminoglycoside gentamicin, have been incorporated into local delivery sponges or hydrogels.73–75 One of the main disadvantages of chitosan sponge based local delivery systems is their dependence upon diffusion, which may result in not all aspects of an implant receiving antibiotics above the MBIC.23,27 Sponges have an advantage of being loaded with a customizable treatment solution immediately prior to application. Sponges do not require prefabrication in comparison with coatings or hydrogels, giving them practicability for quick interventions as needed, whether in the field or on the surgical table.75,76 These results extended those previously obtained that demonstrate biofilm inhibition in soft tissue implant-associated models10 providing credence to activity in prevention of bone infection. One major limitation for this study was the lack of biofilm formation of E. coli in the in vivo model. The strain of E. coli (ATCC 25922) was selected based on published osteomyelitis model, in which the robustness of this strain for biofilm-associated osteomyelitis was not demonstrated.60 In efforts to reduce or prevent severe morbidity and mortality that have previously occurred in polymicrobial models containing S. aureus and P. aeruginosa,10E. coli was selected. Lack of the robust biofilm formation greatly limits the ability to generalize conclusions to polymicrobial infections. Future in vivo investigations of polymicrobial biofilm will be modeled to include a more pathogenic, biofilm-forming Gram-negative bacteria, such as P. aeruginosa with an appropriate inoculum adaptation,7,77,78 and including comparisons to systemic delivery. CONCLUSIONS These preliminary results and with further investigations the ability of ciprofloxacin and rifampin, in combination, to inhibit representative Gram-positive and Gram-negative strains of bacteria in vitro and when released from chitosan sponges in vivo may have clinical potential for infection prevention in extremity trauma. The combination was conclusive at inhibiting S. aureus. Complete clearance or inhibition of contaminating biofilm pathogens may be achieved by incorporating these antibiotics into chitosan sponges. The sponge delivery system with clinician-customizable loading may provide effective infection prevention for military musculoskeletal trauma. Acknowledgements The authors would like acknowledge Dr. Joel D. Bumgardner and Dr. Erno Lindner for their guidance throughout study, Brandico Barr, Daniel Ahn, Logan R. Boles, Lauren Dishmon, and Rukhsana Awais for assistance with various aspects of the study. Presentations Presented as a poster at the 2016 Military Health System Research Symposium, Kissimmee, FL (Abstract number: MHSRS-16-1333). Funding This study was supported by a grant from the Department of Defense (#W81XWH-12-2-0020). Material donation of sponges was provided by Bionova Medical. REFERENCES 1 Logeart-Avramoglou D, Anagnostou F, Bizios R, Petite H: Engineering bone: challenges and obstacles. J Cell Mol Med  2005; 9( 1): 72– 84. Google Scholar CrossRef Search ADS   2 Sahli Z, Bizri A, Abu-Sittah G: Microbiology and risk factors associated with war-related wound infections in the Middle East. Epidemiol Infect  2016; 144( 13): 2848– 57. Google Scholar CrossRef Search ADS   3 Patel A, Calfee RP, Plante M, Fischer SA, Arcand N, Born C: Methicillin-resistant Staphylococcus aureus in orthopaedic surgery. J Bone Jt Surg  2008; 90( B): 1401– 06. Google Scholar CrossRef Search ADS   4 Trampuz A, Widmer AF: Infections associated with orthopedic implants. Curr Opin Infect Dis  2006; 19: 349– 56. Google Scholar CrossRef Search ADS   5 Eardley W, Brown K, Bonner T, Green A, Clasper J: Infection in conflict wounded. Philos Trans R Soc London B Biol Sci  2011; 366( 1562): 204– 18. Google Scholar CrossRef Search ADS   6 Stoodley P, Ehrlich GD, Sedghizadeh PP, et al.  : Orthopaedic biofilm infections. Curr Orthop Pract  2011; 22( 6): 558– 63. Google Scholar CrossRef Search ADS   7 Nana A, Nelson SB, McLaren A, Chen AF: What’s new in Musculoskeletal infection: update on biofilms. J Bone Jt Surg, Am Vol  2016; 98( 14): 1226– 34. Google Scholar CrossRef Search ADS   8 Murray CK: Epidemiology of infections associated with combat-related injuries in Iraq and Afghanistan. J Trauma Acute Care Surg  2008; 64( 3): S232– S38. Google Scholar CrossRef Search ADS   9 Jennings JA: CORR Insights(R): local gentamicin delivery from resorbable viscous hydrogels is therapeutically effective. Clin Orthop Relat Res  2015; 473( 1): 348– 50. Google Scholar CrossRef Search ADS   10 Jennings JA, Beenken KE, Parker AC, et al.  : Polymicrobial biofilm inhibition effects of acetate‐buffered chitosan sponge delivery device. Macromol Biosci  2016; 16( 4): 591– 98. Google Scholar CrossRef Search ADS   11 Parker AC, Rhodes C, Jennings JA, et al.  : Preliminary evaluation of local drug delivery of amphotericin B and in vivo degradation of chitosan and polyethylene glycol blended sponges. J Biomed Mater Res B Appl Biomater  2016; 104( 1): 78– 87. Google Scholar CrossRef Search ADS   12 Smith JK, Bumgardner JD, Courtney HS, Smeltzer MS, Haggard WO: Antibiotic-loaded chitosan film for infection prevention: a preliminary in vitro characterization. J Biomed Mater Res B Appl Biomater  2010; 94( 1): 203– 11. 13 Hospenthal DR, Murray CK, Andersen RC, et al.  : Guidelines for the prevention of infections associated with combat-related injuries: 2011 update: endorsed by the Infectious Diseases Society of America and the Surgical Infection Society. J Trauma Acute Care Surg  2011; 71( 2): S210– S34. Google Scholar CrossRef Search ADS   14 Diefenbeck M, Mückley T, Hofmann GO: Prophylaxis and treatment of implant-related infections by local application of antibiotics. Injury  2006; 37( 2): S95– S104. Google Scholar CrossRef Search ADS   15 Nandi SK, Mukherjee P, Roy S, Kundu B, De Kumar D, Basu D: Local antibiotic delivery systems for the treatment of osteomyelitis – a review. Mater Sci Eng C  2009; 29( 8): 2478– 85. Google Scholar CrossRef Search ADS   16 Branstetter J, Jackson S, Haggard W, Richelsoph K, Wenke J: Locally-administered antibiotics in wounds in a limb. Bone Joint J  2009; 91( 8): 1106– 09. 17 Ingram PR, Lye DC, Tambyah PA, Goh WP, Tam VH, Fisher DA: Risk factors for nephrotoxicity associated with continuous vancomycin infusion in outpatient parenteral antibiotic therapy. J Antimicrob Chemother  2008; 62( 1): 168– 71. Google Scholar CrossRef Search ADS   18 Lopez-Novoa JM, Quiros Y, Vicente L, Morales AI, Lopez-Hernandez FJ: New insights into the mechanism of aminoglycoside nephrotoxicity: an integrative point of view. Kidney Int  2011; 79( 1): 33– 45. Google Scholar CrossRef Search ADS   19 Murillo-Cuesta S, Contreras J, Cediel R, Varela-Nieto I: Comparison of different aminoglycoside antibiotic treatments to refine ototoxicity studies in adult mice. Lab Anim  2010; 44( 2): 124– 31. Google Scholar CrossRef Search ADS   20 Agrawal CM: Introduction to Biomaterials: Basic Theory with Engineering Applications. Cambridge Texts in Biomedical Engineering , Vol. XVI. New York City, C.U. Press, 2014. 21 Robinson D, Alk D, Sandbank J, Farber R, Halperin N: Inflammatory reactions associated with a calcium sulfate bone substitute. Ann Transpl  1998; 4( 3-4): 91– 7. 22 Dash M, Chiellini F, Ottenbrite R, Chiellini E: Chitosan – a versatile semi-synthetic polymer in biomedical applications. Prog Polym Sci  2011; 36( 8): 981– 1014. Google Scholar CrossRef Search ADS   23 Zahedi P, Rezaeian I, Ranaei‐Siadat SO, Jafari SH, Supaphol P: A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages. Polym Adv Technol  2010; 21( 2): 77– 95. 24 Monette A, Ceccaldi C, Assaad E, Lerouge S, Lapointe R: Chitosan thermogels for local expansion and delivery of tumor-specific T lymphocytes towards enhanced cancer immunotherapies. Biomaterials  2016; 75: 237– 49. Google Scholar CrossRef Search ADS   25 Parker AC, Jennings JA, Bumgardner JD, Courtney HS, Lindner E, Haggard WO: Preliminary investigation of crosslinked chitosan sponges for tailorable drug delivery and infection control. J Biomed Mater Res B Appl Biomater  2013; 101( 1): 110– 23. Google Scholar CrossRef Search ADS   26 Haggard WO, Noel SP, Bumgardner JD: Compositions and methods for delivering an agent to a wound. 2015.Available at https://www.google.com/patents/US8993540; accessed March 15, 2017. 27 Noel SP, Courtney HS, Bumgardner JD, Haggard WO: Chitosan sponges to locally deliver amikacin and vancomycin: a pilot in vitro evaluation. Clin Orthop Relat Res  2010; 468( 8): 2074– 80. Google Scholar CrossRef Search ADS   28 Stinner DJ, Noel SP, Haggard WO, Watson JT, Wenke JC: Local antibiotic delivery using tailorable chitosan sponges: the future of infection control? J Orthop Trauma  2010; 24( 9): 592– 97. Google Scholar CrossRef Search ADS   29 Reves BT, Bumgardner JD, Cole JA, Yang Y, Haggard WO: Lyophilization to improve drug delivery for chitosan‐calcium phosphate bone scaffold construct: a preliminary investigation. J Biomed Mater Res B Appl Biomater  2009; 90( 1): 1– 10. Google Scholar CrossRef Search ADS   30 Coiffier G, Albert J-D, Arvieux C, Guggenbuhl P: Optimizing combination rifampin therapy for staphylococcal osteoarticular infections. Joint Bone Spine  2013; 80( 1): 11– 7. Google Scholar CrossRef Search ADS   31 Buensalido JAL, DeMarco CE, Lerner SA: Mechanisms of Action of Antibacterial Agents. In: Practical Handbook of Microbiology . Edited by Goldman E, Green LH. CRC Press, 2015. 32 Drlica K: Mechanism of fluoroquinolone action. Curr Opin Microbiol  1999; 2( 5): 504– 08. Google Scholar CrossRef Search ADS   33 Mitscher LA: Bacterial topoisomerase inhibitors: quinolone and pyridone antibacterial agents. Chem Rev  2005; 105( 2): 559– 92. Google Scholar CrossRef Search ADS   34 Khodursky AB, Cozzarelli NR: The mechanism of inhibition of topoisomerase IV by quinolone antibacterials. J Biol Chem  1998; 273( 42): 27668– 77. Google Scholar CrossRef Search ADS   35 Cheng G, Hao H, Dai M, Liu Z, Yuan Z: Antibacterial action of quinolones: from target to network. Eur J Med Chem  2013; 66: 555– 62. Google Scholar CrossRef Search ADS   36 Bourguignon GJ, Levitt M, Sternglanz R: Studies on the mechanism of action of nalidixic acid. Antimicrob Agents Chemother  1973; 4( 4): 479– 86. Google Scholar CrossRef Search ADS   37 Sugino A, Peebles CL, Kreuzer KN, Cozzarelli NR: Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc Natl Acad Sci USA  1977; 74( 11): 4767– 71. Google Scholar CrossRef Search ADS   38 Emmerson A, Jones A: The quinolones: decades of development and use. J Antimicrob Chemother  2003; 51( suppl 1): 13– 20. Google Scholar CrossRef Search ADS   39 Stein GE, Goldstein EJ: Fluoroquinolones and anaerobes. Clin Infect Dis  2006; 42( 11): 1598– 607. Google Scholar CrossRef Search ADS   40 Moudgal VV, Kaatz GW: Fluoroquinolone resistance in bacteria. In: Antimicrobial Drug Resistance , pp 195– 205. Edited by Mayers DL. Springer, 2009. Google Scholar CrossRef Search ADS   41 Smith JK, Moshref AR, Jennings JA, Courtney HS, Haggard WO: Chitosan sponges for local synergistic infection therapy: a pilot study. Clin Orthop Relat Res  2013; 471( 10): 3158– 64. Google Scholar CrossRef Search ADS   42 Liu J, Sun J, Zhang W, Gao K, He Z: HPLC determination of rifampicin and related compounds in pharmaceuticals using monolithic column. J Pharm Biomed Anal  2008; 46( 2): 405– 09. Google Scholar CrossRef Search ADS   43 Ali SA, Mmuo CC, Abdulraheem RO, et al.  : High performance liquid chromatography (HPLC) Method development and validation indicating assay for ciprofloxacin hydrochloride. J Appl Pharm Sci  2011; 1( 8): 239– 43. 44 Baietto L, D’Avolio A, De Rosa FG, et al.  : Development and validation of a simultaneous extraction procedure for HPLC-MS quantification of daptomycin, amikacin, gentamicin, and rifampicin in human plasma. Anal Bioanal Chem  2010; 396( 2): 791– 98. Google Scholar CrossRef Search ADS   45 Belal FF, El-Din MKS, Eid MI, El-Gamal RM: Micellar HPLC method using monolithic column for the simultaneous determination of linezolid and rifampicin in pharmaceuticals and biological fluids. Analytical Methods  2013; 5( 21): 6165– 76. Google Scholar CrossRef Search ADS   46 Chamseddin C, Jira T: Comparison of the chromatographic behavior of levofloxacin, ciprofloxacin and moxifloxacin on various HPLC phases. . Die Pharmazie – An Int J Pharm Sci  2011; 66( 4): 244– 48. 47 Haeseker M, Verbon A, Welzen J, Neef C, Bruggeman C, Stolk L: A simple and rapid RP-HPLC method to determine ciprofloxacin levels in human serum. Asian J Pharm Biol Res  2011; 1( 3): 350– 54. 48 Hasan N, Siddiqui FA, Sher N, Shafi N, Zubair A, Afzal M: Development and validation of a reverse phase HPLC method for the analysis of ciprofloxacin and its application in bulk and different dosage formulations. World Appl Sci J  2014; 31( 5): 730– 40. 49 Hussain A, Hanif M, Shoiab MH, Yousuf RI, Shafi N: Bioanalytical method development and validation of ciprofloxacin by RP-HPLC method. Asian J Pharm Biol Res  2012; 2( 4): 219– 24. 50 Iqbal Z, Khan J, Khan M, Bilal M, Khan T: The development and validation of HPLC-UV method for analysis of ciprofloxacin in serum and aqueous humour. Arch Pharm Pract  2011; 2( 3): 116– 22. 51 Kulsum S, Reddy CR, Durga MK, Padmalatha M: A simple and validated RP-HPLC method for the simultaneous estimation of tinidazole and ciprofloxacin in bulk and pharmaceutical dosage forms. Int J Res Dev Pharm Life Sci  2012; 2( 1): 238– 43. 52 Pal A, Bawankule DU, Darokar MP, et al.  : Influence of Moringa oleifera on pharmacokinetic disposition of rifampicin using HPLC‐PDA method: a pre‐clinical study. Biomed Chromatogr  2011; 25( 6): 641– 45. Google Scholar CrossRef Search ADS   53 Patel SA: Development and validation of RP-HPLC method for simultaneous determination of Ciprofloxacin and Ornidazole in tablets. Int J Curr Pharm Res  2011; 3( 4): 72– 5. 54 Torres NH, Américo JHP, Nazato C, et al.  : Optimization methodology for detection of antimicrobial ciprofloxacin by HPLC-FLD. Optimization  2012; 4( 2): 59– 62. 55 Beenken KE, Blevins JS, Smeltzer MS: Mutation of sarA in Staphylococcus aureus limits biofilm formation. Infect Immun  2003; 71( 7): 4206– 11. Google Scholar CrossRef Search ADS   56 Masters E, Harris M, Jennings J: Cis-2-decenoic acid interacts with bacterial cell membranes to potentiate additive and synergistic responses against biofilm. J Bacteriol Mycol  2016; 3( 3): 1031– 38. 57 Hall M, Middleton R, Westmacott D: The fractional inhibitory concentration (FIC) index as a measure of synergy. J Antimicrob Chemother  1983; 11( 5): 427– 33. Google Scholar CrossRef Search ADS   58 Bernthal NM, Stavrakis AI, Billi F, et al.  : A mouse model of post-arthroplasty Staphylococcus aureus joint infection to evaluate in vivo the efficacy of antimicrobial implant coatings. PLoS One  2010; 5( 9): e12580. Google Scholar CrossRef Search ADS   59 Smeltzer MS, Thomas JR, Hickmon SG, et al.  : Characterization of a rabbit model of staphylococcal osteomyelitis. J Orthop Res  1997; 15( 3): 414– 21. Google Scholar CrossRef Search ADS   60 Stewart RL, Cox JT, Volgas D, et al.  : The use of a biodegradable, load-bearing scaffold as a carrier for antibiotics in an infected open fracture model. J Orthop Trauma  2010; 24( 9): 587– 91. Google Scholar CrossRef Search ADS   61 Möker N, Dean CR, Tao J: Pseudomonas aeruginosa increases formation of multidrug-tolerant persister cells in response to quorum-sensing signaling molecules. J Bacteriol  2010; 192( 7): 1946– 55. Google Scholar CrossRef Search ADS   62 Breidenstein EB, de la Fuente-Núñez C, Hancock RE: Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol  2011; 19( 8): 419– 26. Google Scholar CrossRef Search ADS   63 Yoon J, Urban C, Terzian C, Mariano N, Rahal JJ: In vitro double and triple synergistic activities of polymyxin B, imipenem, and rifampin against multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother  2004; 48( 3): 753– 57. Google Scholar CrossRef Search ADS   64 Tascini C, Tagliaferri E, Giani T, et al.  : Synergistic activity of colistin plus rifampin against colistin-resistant KPC-producing Klebsiella pneumoniae. Antimicrob Agents Chemother  2013; 57( 8): 3990– 93. Google Scholar CrossRef Search ADS   65 Perlroth J, Kuo M, Tan J, Bayer AS, Miller LG: Adjunctive use of rifampin for the treatment of Staphylococcus aureus infections: a systematic review of the literature. Arch Intern Med  2008; 168( 8): 805– 19. Google Scholar CrossRef Search ADS   66 Bustamante C, Wharton R, Wade J: In vitro activity of ciprofloxacin in combination with ceftazidime, aztreonam, and azlocillin against multiresistant isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother  1990; 34( 9): 1814– 15. Google Scholar CrossRef Search ADS   67 Moody J, Gerding D, Peterson L: Evaluation of ciprofloxacin’s synergism with other agents by multiple in vitro methods. Am J Med  1987; 82( 4A): 44– 54. 68 Zimmerli W, Widmer AF, Blatter M, Frei R, Ochsner PE: Role of rifampin for treatment of orthopedic implant–related staphylococcal infections: a randomized controlled trial. JAMA  1998; 279( 19): 1537– 41. Google Scholar CrossRef Search ADS   69 Henry N, Rouse M, Whitesell A, McConnell M, Wilson W: Treatment of methicillin-resistant Staphylococcus aureus experimental osteomyelitis with ciprofloxacin or vancomycin alone or in combination with rifampin. Am J Med  1987; 82( 4A): 73– 5. 70 Widmer AF, Gaechter A, Ochsner PE, Zimmerli W: Antimicrobial treatment of orthopedic implant-related infections with rifampin combinations. Clin Infect Dis  1992; 14( 6): 1251– 53. Google Scholar CrossRef Search ADS   71 Jennings JA, Carpenter DP, Troxel KS, et al.  : Novel antibiotic-loaded point-of-care implant coating inhibits biofilm. Clin Orthop Relat Res  2015; 473( 7): 2270– 82. Google Scholar CrossRef Search ADS   72 Berretta JM, Jennings JA, Courtney HS, Beenken KE, Smeltzer MS, Haggard WO: Blended chitosan paste for infection prevention: preliminary and preclinical evaluations. Clin Orthop Relat Res  2017; 475: 1857– 1870. Google Scholar CrossRef Search ADS   73 Chang WK, Srinivasa S, MacCormick AD, Hill AG: Gentamicin-collagen implants to reduce surgical site infection: systematic review and meta-analysis of randomized trials. Ann surg  2013; 258( 1): 59– 65. LWW. Google Scholar CrossRef Search ADS   74 Penn-Barwell JG, Murray CK, Wenke JC: Local antibiotic delivery by a bioabsorbable gel is superior to PMMA bead depot in reducing infection in an open fracture model. J Orthop Trauma  2014; 28( 6): 370– 75. Google Scholar CrossRef Search ADS   75 Overstreet D, McLaren A, Calara F, Vernon B, McLemore R: Local gentamicin delivery from resorbable viscous hydrogels is therapeutically effective. Clin Orthop Relat Res  2015; 473( 1): 337– 47. Google Scholar CrossRef Search ADS   76 Yeo Y, Highley CB, Bellas E, et al.  : In situ cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model. Biomaterials  2006; 27( 27): 4698– 705. Google Scholar CrossRef Search ADS   77 Dalton T, Dowd SE, Wolcott R, et al.  : An in vivo polymicrobial biofilm wound infection model to study interspecies interactions. PLoS One  2011; 6( 11): e27317. Google Scholar CrossRef Search ADS   78 Dowd SE, Sun Y, Secor PR, et al.  : Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiol  2008; 8( 1): 43. Google Scholar CrossRef Search ADS   Author notes The views expressed in this paper are those of the authors and do not necessarily represent the official position or policy of the U.S. Government of the Department of Defense. © Association of Military Surgeons of the United States 2018. All rights reserved. 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TI - Ciprofloxacin and Rifampin Dual Antibiotic-Loaded Biopolymer Chitosan Sponge for Bacterial Inhibition
JF - Military Medicine
DO - 10.1093/milmed/usx150
DA - 2018-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/ciprofloxacin-and-rifampin-dual-antibiotic-loaded-biopolymer-chitosan-r34OXyXx0T
SP - 433
EP - 444
VL - 183
IS - suppl_1
DP - DeepDyve
ER -