TY - JOUR AU1 - BS, Logan R Boles, AU2 - MS, Rukhsana Awais, AU3 - PhD, Karen E Beenken, AU4 - PhD, Mark S Smeltzer, AU5 - PhD, Warren O Haggard, AU6 - PhD, Amber Jennings Jessica, AB - Abstract Military personnel have high risk for infection, particularly those with combat-related extremity trauma. Administration of multiple or broad-spectrum antibiotics provides clinicians with a strategy for preventing biofilm-based medical device infections. Selection of effective antibiotic combinations based on common pathogens may be used to improve chitosan wound dressing sponge-based local antibiotic delivery systems. In vitro assays in this study demonstrate that vancomycin and amikacin have a synergistic relationship against a strain of osteomyelitis-producing Gram-positive Staphylococcus aureus, although an indifferent relationship was observed against Gram-negative Pseudomonas aeruginosa. In an in vivo model of orthopedic hardware-associated polymicrobial (S. aureus and Escherichia coli) biofilm, chitosan sponges loaded with a combination of vancomycin and amikacin at 5 mg/mL each showed a greater percentage of complete clearance, 50%, than either antibiotic alone, 8.33%. Doubling the loading concentration of the combination achieved a complete clearance rate of 100%, a four log-fold reduction of S. aureus on the wire and a six log-fold reduction in bone. E. coli was detected in bone of untreated animals but did not form biofilm on wires. Results demonstrate the clinical potential of chitosan sponges to prevent infection and illustrates antibiotic selection and loading concentrations necessary for effective biofilm prevention. INTRODUCTION Despite advancements in medical treatment, wound infection is the most significant cause of morbidity and mortality in U.S. Military service members who survive combat-related injuries.1 Studies of war-related blast injury wound infection report several contributing factors: pathogen, injury type and severity, study population, infection outcome, and geographic zones. A recent review found war-related infection rates ranging from 4.9% to 78% in Middle Eastern conflict zones.2 These studies included U.S. Military personnel, non-U.S. Military personnel, civilians, and non-blast-related injuries. If civilians and non-U.S. Military personnel are removed, U.S. Military personnel have war-related infection rates ranging from 5.5% to 49%.2 Blast injuries are particularly prone to infection due to the complex geometry of wounds and the presence of environmental contaminants, with some studies showing biofilm forming on similar injuries within a matter of hours.3 More than 65% of infections being treated in the developed world are caused by biofilm forming bacteria.4Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are the most prevalent Gram-positive and Gram-negative pathogens, respectively; they account for up to 75% of biofilm infections associated with medical devices and have traditionally complicated battlefield injuries.3,5 During Operation Iraqi Freedom, Escherichia coli and P. aeruginosa were each found to be contaminating 14% of wounds.5 The presence of one of these bacteria produces a favorable environment for other pathogens and can cause local and/or systemic disease, whereas multiple bacterial strains can develop mutualistically, increasing the severity of infection and exacerbating symptomatology.6,7 This innate synergism between microorganisms in polymicrobial infections makes treatment difficult, leading to delayed wound healing and increased morbidity.8 Infection treatment can increase hospitalization costs up to 300%, prevents military personnel from returning to their position, increases risk of repeated hospitalization, and may permanently incapacitate them.5,9–11 Prevention of infection after an injury is a key strategy in managing combat-related trauma, with standard of care being administration of systemic antibiotics within 3 h of sustaining injury to lower the chance of wound infection and bacterial colonization.3,9 Local delivery systems for antibiotics, such as antibiotic-loaded bone cement and calcium sulfate, are growing in popularity as an adjunctive strategy to systemic delivery.12–14 Local delivery systems are advantageous over systemic administration because they deliver high concentrations of antibiotic directly to the affected tissue12 and minimize risk of toxicity to organs.13,15–17 Each of these systems have been used successfully albeit with some drawbacks. Poly(methyl methacrylate) (PMMA) is non-degradable and requires a secondary surgery to remove the implant for complete wound healing. Also, once the PMMA has released its antibiotic load, it serves as a surface for bacteria to adhere to and form a biofilm.18 Rapid resorption of calcium sulfate pellets results in calcium-rich fluid that incites an inflammatory response.19 To account for the possibility of polymicrobial contamination, the ability for a clinician to load a local delivery device with broad-spectrum or multiple antibiotics at the time of intervention would be advantageous. Therefore, desirable characteristics of a local antibiotic delivery system include extended release profile, biocompatibility, complete biodegradability into biocompatible degradation products, and tailored antimicrobial selection.20 Chitosan, a versatile natural biopolymer, has been used in the fabrication of many types of drug delivery systems,20–22 including lyophilized sponges.23 Commercially available chitosan wound dressings in the form of a sponge were approved by the Food and Drug Administration (FDA) for wound management and may be hydrated with saline for use with traumatic injury sites, surgical wounds, trauma wounds, and other applications. Physicians may, in the practice of medicine, choose to include antibiotics in the hydrating saline solution for the management of contaminated or infected traumatic injuries.24 The inclusion of antibiotics in the hydrating solution is not specifically cleared by the FDA. Chitosan sponges have shown excellent biocompatibility, degradability, and may be loaded with multiple or broad-spectrum antibiotics by passive absorption and diffusion.20,23–26 Sponges can be manufactured with varying thicknesses and pore size using a lyophilization process to tailor release of antibiotic solutions,23,27 and their geometry can be customized for appropriate coverage of complex wounds. Complex wounds produced by high-energy trauma typically require hardware or medical devices to promote healing. Exposed bone and medical devices are conducive substrates for bacterial attachment and biofilm formation. Treating and preventing biofilm-based medical device infections may require high antibiotic concentrations and may be assisted by anti-biofilm agents.28,29 Delivery of these high concentrations of antibiotic or anti-biofilm molecules may be facilitated through biomaterial local drug delivery systems. Antimicrobials delivered directly to the affected tissue may eliminate microorganisms before biofilm is established. This study seeks to investigate the effects of combining amikacin and vancomycin for local delivery from chitosan sponges for infection prevention. In vitro microbiological assays were used to determine if these antibiotics have additive or synergistic effects on each other against two representative pathogens, S. aureus and P. aeruginosa. A murine model of implant-associated orthopedic infection was then used to determine effects of antibiotic-loaded sponges and loading concentrations on infection prevention. MATERIALS AND METHODS In vitro and in vivo tests were used to evaluate the efficacy of combined therapy of vancomycin and amikacin against pathogens commonly associated with implant-related infection. In Vitro Synergy Assay Checkerboard synergy testing was performed using 96-well microtiter plates. Vancomycin (MP Biomedicals, Santa Ana, CA) and amikacin (MP Biomedicals, Santa Ana, CA) were dissolved in phosphate-buffered saline (PBS) and diluted in tryptic soy broth (TSB) in seven two-fold dilutions for final concentrations of 0–8 μg/mL for vancomycin and 0–128 μg/mL for amikacin in each well. 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; ATCC 49230) or P. aeruginosa (PA ATCC 27317) for a final concentration of approximately 1 × 104 CFUs per well. Plates were incubated for 24 h at 37°C. All bacterial strains used were clinical isolates. UAMS-1 was cultured from the bone of a patient suffering from osteomyelitis and has been shown to form biofilm in vitro and in vivo;30 PA ATCC 27317 isolated from blood is capable of forming biofilm in vitro.31 Planktonic bacteria were removed from the wells by aspirating the liquid without disturbing the biofilm and gently washing with PBS three times. Biofilm bacteria were heat fixed by heating the plate to 60°C for 1 h. Once the biofilm was fixed, it was stained using 100 μL of crystal violet solution. Crystal violet not taken up by the biofilm was removed by carefully rinsing with water. Then, a destaining solution composed of 7.5% acetic acid, 10% methanol, and water was used to dissolve the bound crystal violet. Finally, absorbance measurements were obtained at 540 nm using a plate reader spectrophotometer (Biotek ELx800, Winooski, VT). The amount of antibiotic required to prevent biofilm from forming on a surface is the minimum biofilm inhibitory concentration (MBIC). To quantify relationships between antibiotics, the fractional inhibitory concentration index (FICI) was used.31,32 To determine FICI for each antibiotic, the MBIC for the antibiotic used in combination was divided by the MBIC of each antibiotic alone. Then, the FICI for each antibiotic was summed to acquire a final FICI value. FICI values less than 1 were considered synergistic, between 1 and 2 were additive, equal to 2 were indifferent, and greater than 2 were antagonistic. Chitosan Sponges All chitosan sponges used for experiments were commercially available Sentrex BioSponge (Bionova Medical, Memphis, TN). To prepare hydrated sponges for the in vivo study, sponges were cut into 6-mm coupons and soaked in an antibiotic hydrating solution at the desired concentration for 1 min immediately before application to the wound site. 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 study of orthopedic implant-associated infection33 was adapted to use a polymicrobial mixture of known biofilm forming bacteria: S. aureus (UAMS-1)34 and E. coli (ATCC 25922).35,36 Eight- to twelve-wk-old C57BL/6 mice weighing between 16 and 18 g were anesthetized with isoflurane and avertin (0.4–0.6 mg/g). An incision was made over the left knee, and a hole was drilled into the left distal femur using a 26-gauge syringe needle and then a 23-gauge syringe needle. A sterile 1-cm × 0.6-mm diameter stainless steel Kirschner wire was inoculated with approximately 104 colony forming units (CFUs) of UAMS-1 and 102 CFUs of ATCC 25922 and inserted into the femur as shown in Figure 1. Chitosan sponges loaded with saline (negative control), 5 mg/mL vancomycin, 5 mg/mL amikacin, or a combination of amikacin and vancomycin at 5 mg/mL each were implanted adjacent to the contaminated implant (n = 12 per group). Mice were humanely euthanized after 1 wk, at which time the wire implant and appropriate femur tissue were removed to determine the number of viable bacterial CFUs remaining on/in each. To determine the number of bacterial colonies associated with the implanted device, wire implants were carefully rinsed in triplicate with PBS to remove planktonic bacteria. Afterward, the wire implants were sonicated and vortexed in sterilized PBS, and bone tissue was homogenized and suspended in sterilized PBS. Then, PBS was plated on Tryptic Soy Agar, and the number of colonies were quantified after being incubated overnight at 37°C. Bacterial clearance was accomplished by eliminating bacteria before biofilm could be established on the surface of the implant and defined as an apparent bacterial CFU count of zero. Figure 1. View largeDownload slide Diagram and photographs of polymicrobial murine model of implant-associated infection. Figure 1. View largeDownload slide Diagram and photographs of polymicrobial murine model of implant-associated infection. In a follow-up study, the concentration of E. coli was increased 10-fold, and the treatment groups were narrowed to the vancomycin and amikacin combination and PBS-loaded sponges (n = 12 per group). Antibiotic concentrations were increased to 10 mg/mL based on preliminary findings of synergistic responses. Statistical Analysis 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’s exact test of contingency tables was used to compare the rate of clearance for implants in antibiotic-loaded groups with controls, with Bonferroni post hoc correction in cases of multiple comparisons. RESULTS Synergy Assay Results from the synergy assay showed a synergistic effect between vancomycin and amikacin for prevention of S. aureus (UAMS-1) biofilms with a FICI value of 0.75 (Table I). No additive or synergistic effects were detected between vancomycin and amikacin for prevention of P. aeruginosa (PA ATCC 27317) biofilms as quantified by a FICI value of 2.0 (Table I). Table I. FICI Values and Interpretation for Vancomycin and Amikacin Against UAMS-1 and PA ATCC 27317 Bacteria  Amikacin MBIC (Alone, μg/mL)  Amikacin MBIC (Combo, μg/mL)  Vancomycin MBIC (Alone, μg/mL)  Vancomycin MBIC (Combo, μg/mL)  FICI  Combined Effect  S. aureus (UAMS-1)  16  4  0.125  0.0625  0.75  Synergistic  P. aeruginosa (PA ATCC 27317)  16  16  >64  >64  2  Indifferent  Bacteria  Amikacin MBIC (Alone, μg/mL)  Amikacin MBIC (Combo, μg/mL)  Vancomycin MBIC (Alone, μg/mL)  Vancomycin MBIC (Combo, μg/mL)  FICI  Combined Effect  S. aureus (UAMS-1)  16  4  0.125  0.0625  0.75  Synergistic  P. aeruginosa (PA ATCC 27317)  16  16  >64  >64  2  Indifferent  FICI, fractional inhibitory concentration index; MBIC, minimum biofilm inhibitory concentration; μg/mL, micrograms per milliliter. Table I. FICI Values and Interpretation for Vancomycin and Amikacin Against UAMS-1 and PA ATCC 27317 Bacteria  Amikacin MBIC (Alone, μg/mL)  Amikacin MBIC (Combo, μg/mL)  Vancomycin MBIC (Alone, μg/mL)  Vancomycin MBIC (Combo, μg/mL)  FICI  Combined Effect  S. aureus (UAMS-1)  16  4  0.125  0.0625  0.75  Synergistic  P. aeruginosa (PA ATCC 27317)  16  16  >64  >64  2  Indifferent  Bacteria  Amikacin MBIC (Alone, μg/mL)  Amikacin MBIC (Combo, μg/mL)  Vancomycin MBIC (Alone, μg/mL)  Vancomycin MBIC (Combo, μg/mL)  FICI  Combined Effect  S. aureus (UAMS-1)  16  4  0.125  0.0625  0.75  Synergistic  P. aeruginosa (PA ATCC 27317)  16  16  >64  >64  2  Indifferent  FICI, fractional inhibitory concentration index; MBIC, minimum biofilm inhibitory concentration; μg/mL, micrograms per milliliter. In Vivo Implant-Associated Biofilm Model (5 mg/mL) Chitosan sponges loaded with amikacin or a combination of vancomycin and amikacin significantly reduced CFUs of S. aureus on the surface of the implant (Fig. 2) and in bone (Fig. 3) compared with PBS and vancomycin-loaded sponges. Rates of bacterial clearance on the wire and in bone tissue were statistically higher than PBS-loaded controls in the combination of amikacin and vancomycin group (Fisher’s exact, p = 0.036 and 0.042, respectively), but the corrected p-value considering multiple comparisons against the controls of vancomycin only and amikacin only is not considered statistically significant (p = 0.11). However, complete clearance of bacterial colonies was not achieved for any of the groups. In initial studies, no E. coli colonies were present after 7 d, either in bone or attached to the implant. Subsequent studies increased the inoculum of E. coli by 10-fold to attempt to address this low bacterial growth. Figure 2. View largeDownload slide Data points represent (A) S. aureus colony forming units (CFU) count for individual animals and (B) percent of animals with zero CFU counts (complete clearance). Asterisks represent statistical significance detected between group and phosphate-buffered saline (PBS) controls in two-way ANOVA or Fisher’s exact tests. *p < 0.05; **p < 0.01. Figure 2. View largeDownload slide Data points represent (A) S. aureus colony forming units (CFU) count for individual animals and (B) percent of animals with zero CFU counts (complete clearance). Asterisks represent statistical significance detected between group and phosphate-buffered saline (PBS) controls in two-way ANOVA or Fisher’s exact tests. *p < 0.05; **p < 0.01. Figure 3. View largeDownload slide Data points represent (A) S. aureus colony forming units (CFU) count in bone tissue for individual animals and (B) percent of animals with zero CFU count (complete clearance). Asterisks represent statistical significance detected between group and phosphate-buffered saline (PBS) controls in two-way ANOVA or Fisher’s exact tests. *p < 0.05; **p < 0.01. Figure 3. View largeDownload slide Data points represent (A) S. aureus colony forming units (CFU) count in bone tissue for individual animals and (B) percent of animals with zero CFU count (complete clearance). Asterisks represent statistical significance detected between group and phosphate-buffered saline (PBS) controls in two-way ANOVA or Fisher’s exact tests. *p < 0.05; **p < 0.01. In Vivo Implant-Associated Biofilm Model (10 mg/mL) Although some E. coli colonies were retrieved from bone tissue in negative saline controls, there were no viable colonies after 7 d on the implant (Fig. 4). S. aureus was completely inhibited on the surface of the implant and bone at 7 d using a combination of 10 mg/mL amikacin and 10 mg/mL vancomycin (100% clearance, p < 0.001; Fig. 5). Figure 4. View largeDownload slide Data points represent E. coli colony forming units (CFU) count for individual animals in the increased dose study for (A) wires and (B) Bone. (Note: PBS, phosphate-buffered saline). Figure 4. View largeDownload slide Data points represent E. coli colony forming units (CFU) count for individual animals in the increased dose study for (A) wires and (B) Bone. (Note: PBS, phosphate-buffered saline). Figure 5. View largeDownload slide Data points represent S. aureus CFU count for individual animals in the increased dose study for (A) wires and (B) bone. Asterisks represent statistical significance detected between group and PBS controls in two-way ANOVA. *p < 0.05. Figure 5. View largeDownload slide Data points represent S. aureus CFU count for individual animals in the increased dose study for (A) wires and (B) bone. Asterisks represent statistical significance detected between group and PBS controls in two-way ANOVA. *p < 0.05. DISCUSSION Our study sought to determine if local drug delivery from chitosan sponges could prevent polymicrobial implant-associated biofilm formation. The efficacy of this treatment method was evaluated using a known model of implant infection. Results from this experiment point toward a synergistic relationship against Gram-positive bacteria between vancomycin and amikacin due to complete bacterial clearance of S. aureus after addition of combination-loaded sponges at sufficient concentrations. These results confirm results from previous studies and may be due to antibiotic mechanisms and sites of action.37,38 Vancomycin, a glycopeptide antibiotic, offers coverage against Gram-positive bacteria through inhibition of synthesis of bacterial cell wall phospholipids and preventing peptidoglycan cross-linking.39 On the other hand, amikacin, an aminoglycoside antibiotic, shows activity against Gram-positive and Gram-negative bacteria.39 This class of antibiotics works by diffusing through the outer membrane of the bacteria via porin channels and binding the 30S ribosomal subunit before the ribosome can form.39 There should be an innate synergism between these two classes of antibiotics; the glycopeptide should weaken the cell wall, which will allow the aminoglycoside to more easily access and penetrate the cell membrane. Several studies have observed synergism between vancomycin and aminoglycoside antibiotics against various strains of bacteria such as Enterococci, E. coli, and S. aureus.37,38,40,41 Climo et al. showed an enhancement of in vitro activity of vancomycin with a combination of either tobramycin or gentamicin, while showing no beneficial effect with rifampin, an antimycobacterial.40 Harwick et al. tested vancomycin in combination with and without gentamicin against Enterococci, a Gram-positive bacteria.41 This study showed that vancomycin alone showed significant inhibitory effects but reduced bactericidal activity; using the combination with gentamicin, vancomycin displayed a substantial increase in bactericidal activity.41 This study confirms that this synergistic relationship between antibiotics is active against a strain of S. aureus isolated from an osteomyelitic VA patient in a VA hospital.42 Little comparative information was found in the literature for synergism between vancomycin and amikacin. The synergism between amikacin and vancomycin may be the reason the combination of both produced a higher rate of clearance in the initial 5 mg/mL loading study. Even with a reduction of CFUs, remaining bacteria could resurge and result in continued infection.3,25 Recently, two similar studies obtained complete bacterial clearance of P. aeruginosa and S. aureus using a phospholipid coating impregnated with amikacin and vancomycin43 or injectable hydrogels containing high doses of gentamicin.44 This highlights a disadvantage of the chitosan sponge-based delivery system in that drug release is dependent on diffusion and may lead to portions of the implant not receiving antibiotics above the MBIC.23,26 The trade-off is that sponges can be loaded immediately before treatment and do not require prefabrication like coatings or hydrogels, making them practical for quick intervention during field care or initial surgical interventions.44,45 Also, these studies both used a higher loading concentration of antibiotics than the initial study, which correlates with our results of complete clearance after increased loading concentration. Concentrations of 10 mg/mL or higher can be mixed from available intravenous drip containers with antibiotic in powdered form. Our results extend the results of previous studies showing biofilm inhibition in soft tissue implant-associated models25 to confirm activity in prevention of bone infection. A limitation of this preliminary study was the use of a single in vitro preventative model to determine the efficacy of the antimicrobial combinations. The checkerboard assay is a static model of biofilm growth and does not fully mimic conditions within the body. However, this assay is an effective screening method for determining the MBIC of the antimicrobial combinations.46,47 To more closely model natural biofilm conditions, a flow model for biofilm growth may be employed instead of a static method.48In vitro models are invaluable for initial experimentation, but they lack the complexity to accurately represent clinical biofilms. Another limitation is that the polymicrobial biofilm was composed of only two bacterial species. Clinical studies of biofilms show that their composition may consist of several colocalized bacterial species and fungi.6,7 However, co-culturing microorganisms that are present in natural biofilms is difficult and usually results in one of the co-cultured microorganisms diminishing or eliminating the other species before polymicrobial biofilms can form.49 In continuing research and future studies, limitations will be addressed by refining in vitro and in vivo models to more closely model clinical polymicrobial biofilms. Another limitation for the in vivo model was the strain of E. coli used (ATCC 25922), which was found to be less robust than expected and did not readily form biofilm on the pin. This method was adapted from an osteomyelitis model, and the previous investigators also reported issues with this particular strain.35E. coli was selected to avoid severe complications in animals, such as morbidity and mortality, that have occurred previously in polymicrobial models combining S. aureus and P. aeruginosa.25 The lack of a robust polymicrobial biofilm-based medical device infection in the in vivo model does limit the generalizability of these results to the prevention of polymicrobial biofilm. For future in vivo studies, adaptation of inoculum to include a more pathogenic, biofilm forming Gram-negative bacteria, such as P. aeruginosa, may be used.3,50,51 Another addition to future evaluations will be the addition of a control group that contains mice treated with systemic antibiotic administration. CONCLUSIONS The local delivery strategy for infection prevention in extremity trauma was effective. Chitosan sponges loaded with a combination of amikacin and vancomycin inhibited both S. aureus and E. coli in this model of biofilm-based medical device infection. This success and associated in vitro testing demonstrate the clinical potential for this local delivery strategy. Inhibition of biofilm formation and clearance of the biofilm forming pathogens on the implanted wires were achieved through increasing the antibiotic loading of the sponge delivery device. Acknowledgments The authors would like to acknowledge assistance from Dr. Harry Courtney; Dr. Joel Bumgardner for assistance with study design; Carlos Wells for research assistance; and Lauren Dishmon, Chris Alexander, and Mike Harris for synergism help. Funding This study was supported by a grant from the Department of Defense (grant no. W81XWH-12-2-0020). Material donation of sponge was provided by Bionova Medical. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) TI - Local Delivery of Amikacin and Vancomycin from Chitosan Sponges Prevent Polymicrobial Implant-Associated Biofilm JF - Military Medicine DO - 10.1093/milmed/usx161 DA - 2018-03-01 UR - https://www.deepdyve.com/lp/oxford-university-press/local-delivery-of-amikacin-and-vancomycin-from-chitosan-sponges-5CSeMymMBf SP - 459 EP - 465 VL - 183 IS - suppl_1 DP - DeepDyve ER -