A Rabbit Osteomyelitis Model to Simulate Multibacterial War Wound Infections

MILITARY MEDICINE, 178, 6:696, 2013 A Rabbit Osteomyelitis Model to Simulate Multibacterial War Wound Infections Li-Yan Yin, MD; Maurice M. Manring, PhD; Jason H. Calhoun, MD, FACS ABSTRACT A challenge facing military caregivers is the presence of multidrug-resistant infection in extremity wounds. Most frequently identified resistant strains are methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB). We adapted an existing osteomyelitis model to simulate an infected extremity wound for antibiotic testing. New Zealand White Rabbits (n = 95) were divided into 6 inoculation groups for infection with MRSA, KP, PA, and AB alone, and in multibacteria infections. Sodium morrhuate was injected into the left tibia to simulate blast wound trauma, then the respective bacteria or combination of pathogens, and finally sterile saline were injected. Colony-forming units for the mono-organism groups showed that AB, KP, or PA alone at 10 7 colony-forming units per ml (CFUs/mL) was effective for rabbit osteomyelitis induction. Colonyforming units for the multiorganism groups showed that the combination of AB (10 7 CFUs/mL)/KP (10 7 CFUs/mL)/PA (10 7 CFUs/mL)/MRSA (10 5 CFUs/mL) yielded a 100% osteomyelitis induction rate. At 8 weeks, however, only one mono-bacterial group and one multibacterial group showed significant radiographic improvement ( p < 0.05). The rabbit model of osteomyelitis can be adapted to study infected blast wounds typical of those seen in veterans. To our knowledge, this is the first demonstration of the model simulating multibacterial infections with multidrug-resistant organisms. INTRODUCTION The rabbit model for osteomyelitis, which has been used for decades and modified by dozens of researchers, has proved its effectiveness in the testing of novel antibiotics and therapy combinations to provide culture-directed treatment for infections. 1,2 However, the model has not been adapted to study the complications (multiple, resistant bacterial strains and wound necrosis) associated with trauma in general, or blast wounds in particular. In Afghanistan and Iraq, the military has an excellent record of providing rapid and highly advanced care aimed at fighting infection. However, a decrease in mortality rates because of the use of improved body armor and rapid evacuation in treatment has been accompanied by a substantial risk of developing infections with multidrug-resistant organisms (MDROs). One of the major challenges facing U.S. military caregivers is the presence of MDROs in orthopedic extremity wounds. 3,4 The most frequently identified resistant strains of bacteria in military and civilian hospitals are methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (PA), Klebsiella pneumonia (KP), and Acinetobacter baumannii (AB). 5 9 We sought to advance the rabbit osteomyelitis model by using more relevant bacterial strains (AB, PA, KP, and MRSA) alone or in combinations and in a multi-infection model, all in the presence of wound necrosis. The study we Department of Orthopaedic Surgery, The Ohio State University Medical Center, 725 Prior Hall, 376 west 10th avenue, Columbus, OH 43210. All institutional and national guidelines for the care and use of laboratory animals were followed. All methods were approved and monitored for compliance to national standards by our institution s Animal Use and Care CommitteeandbytheAnimalCareand Use Review Office (ACURO), U.S. Army Medical Research and Materiel Command. doi: 10.7205/MILMED-D-12-00550 proposed will suggest to researchers a new pathway to study the antibiotic treatment of infection in necrotic wounds seen not only in battlefield environments but in other civilian trauma as well. MATERIALS AND METHODS All methods were approved and monitored for compliance to national standards by our institution s Animal Use and Care Committee and by the Animal Care and Use Review Office, U.S. Army Medical Research and Materiel Command. New Zealand White rabbits, 8 to 12 weeks old and weighing 2.0 to 3.5 kg, were used for this study. Strains of each pathogen were obtained from cultures of wounded veterans treated at Brooke Army Medical Center, Fort Sam Houston, Texas. After the rabbits underwent a minimum 7-day period of acclimatization, an 18-gauge needle was inserted percutaneously through the lateral aspect of the left tibial metaphysis into the intramedullary cavity under anesthesia, then 0.15 ml sodium morrhuate, infection bacteria (KP, AB, PA, and MRSA), and sterile saline were injected sequentially in five groups of 15 rabbits and one group of 20, organized into a total of 19 subgroups (n = 5) to simulate mono- and multiorganism infection (Table I). In the final group of rabbits (group 6), which was injected with all four pathogens, the dosage of sodium morrhuate was varied from 0.15 to 0.25 ml among the three subgroups to simulate different levels of damage to the tibia. Radiographs of tibias were taken at 2, 6, and 8 weeks. Rabbits were anesthetized with 0.6 mg PromACE/ ketamine (45 mg/kg) by subcutaneous injection before radiological testing. Radiographs were scored on a scale of 0 to 4+ by three investigators and the scores averaged (Fig. 1, Table II), a system we have described in previous studies of antibiotics. 10 12 The rabbits with radiographic scored 696

TABLE I. Rabbits Were Divided into Six Groups of 15 Animals Apiece Infection rate 8 weeks Groups Inoculation Titer (CFUs/mL) Marrow (Mean) Marrow (SD) Bone (Mean) Bone (SD) (%) 1 A AB (1 +10 5 )0.00E + 00 0.00E + 00 0.00E + 00 0.00E + 00 0.00 B AB (1 +10 7 )5.72E + 01 7.04E + 01 0.00E + 00 0.00E + 00 40.00 C AB (1 +10 8 )1.38E + 03 2.55E + 03 1.89E + 01 3.78E + 01 60.00 2 A PA (1 +10 5 )0.00E + 00 0.00E + 00 0.00E + 00 0.00E + 00 0.00 B PA (1 +10 7 )3.42E + 04 6.52E + 04 3.73E + 03 4.54E + 03 100.00 C PA (1 +10 8 )1.90E + 02 3.50E + 02 3.65E + 02 5.86E + 02 80.00 3 A KP (1 +10 5 )6.67E + 03 1.33E + 04 6.34E + 01 3.39E + 01 40.00 B KP ( 1 +10 7 )2.08E + 04 3.87E + 04 8.59E + 03 1.23E + 04 80.00 C KP (1 +10 8 )3.92E + 02 7.84E + 02 4.45E + 03 7.59E + 03 80.00 4 A AB (1 +10 8 ) + 0.00E + 00 0.00E + 00 0.00E + 00 0.00E + 000.00 B PA (1 +10 7 ) + 2.17E + 02 4.35E + 02 0.00E + 00 0.00E + 0020.00 C KP (1 +10 7 ) + 2.51E + 03 5.01E + 03 1.57E + 04 2.18E + 0460.00 5 A AB (1 +10 7 ) + PA (1 +10 7 ) + 2.82E + 04 5.10E + 04 4.18E + 05 8.19E + 05 100.00 B PA (1x10 7 ) + KP (1x10 7 ) + 1.12E + 04 2.18E + 04 7.50E + 04 9.41E + 04 60.00 C KP (1 +10 7 ) + AB (1 +10 7 ) + 6.13E + 05 1.23E + 06 1.12E + 07 2.23E + 07 40.00 D KP (1 +10 7 ) + AB (1 +10 7 ) + 2.83E + 02 4.64E + 02 5.17E + 05 9.04E + 05 100.00 PA (1 +10 7 ) 6 A KP (1 +10 7 ) + AB (1 +10 7 ) + 1.01E + 07 2.03E + 07 7.33E + 07 1.47E + 07 40.00 PA (1 +10 7 ) + : 0.15 ml Sodium Morrhuate B KP (1 +10 7 ) + AB (1 +10 7 ) + 4.24E + 02 6.33E + 02 1.48E + 05 2.15E + 05 100.00 PA (1 +10 7 ) + : 0.2 ml Sodium Morrhuate C KP (1 +10 7 ) + AB (1 +10 7 ) + 1.77E + 06 3.55E + 06 5.18E + 06 1.01E + 07 60.00 PA (1 +10 7 ) + : 0.25 ml Sodium Morrhuate Groups 1 to 3, simulated mono-bacterial infections. Groups 4 to 6, simulated multibacterial infections. Three subgroups (n = 5) were created in each group to test different inoculation levels (Groups 1 5) or to experiment with different concentrations of sodium morrhuate (Group 6). above Grade 2 were considered successful in induction of osteomyelitis. Rabbits were euthanized at 8 weeks. Left tibias were harvested from all rabbits. Concentrations of bacteria in bone matrix and bone marrow were determined. Marrow and the intramedullary canal of bilateral tibiae were swabbed with sterile cotton-tip applicators; inoculated applicators were streaked onto plates of Trypticase Soy Agar II supplemented with 5% (v/v) defibrinated sheep blood (BBL, Becton, Dickinson & Co., Sparks, Maryland) and then placed into tubes containing 5 ml of Trypticase Soy Broth (BBL). Plates and tubes were incubated at 37 C for 24 hours, and the presence or absence of growth in both media was recorded. Marrow from each rabbit tibia was deposited into sterile 50-mL centrifuge tubes and weighed. Matrix from each rabbit tibia was cut into 0.5 cm 2 chips, placed in sterile 50-mL centrifuge tubes, and weighed. Physiological saline was added at a ratio of 3 ml of saline per gram FIGURE 1. Representative lateral X-rays of Grades 0 to 4 in radiographic scoring system, with Grade 0 representing no infection and Grade 4 representing highest level of infection. Each representative image of a left tibia received unanimous score from graders: A, Grade 0 (normal); B, Grade 1, shows elevation of periosteum (arrow); C, Grade 2, <10% disruption of normal bone architecture (arrow); D, Grade 3, 10 to 40% disruption of bone architecture (arrow); E, Grade 4, >40% disruption, entire tibia compromised (circle). 697

TABLE II. Radiographic Grading Grade a Description of Changes 0 Normal, No Change Compared With Right Tibia 1+ Elevation or Disruption of Periosteum, or Both; Soft Tissue Swelling 2+ <10% Disruption of Normal Bone Architecture 3+ 10 40% Disruption of Normal Bone Architecture 4+ >40% Disruption of Normal Bone Architecture a Visually estimated percentage of disrupted bone. TABLE III. Radiographic Scores for the Mono-Bacterial War Wound Groups Demonstrating That Only One Group (2A) Showed Significant Improvement at 8 Weeks Groups Inoculation Titer (CFUs/mL) 2 weeks 6 weeks 8 weeks 1 A AB (1 +10 5 ) 0.8 ± 0.7 1.5 ± 1.1 1.0 ± 1.3 B AB (1 +10 7 ) 0.9 ± 0.5 1.1 ± 0.6 0.3 ± 0.4 C AB (1 +10 8 ) 1.7 ± 1.0 1.1 ± 0.6 0.8 ± 0.7 2 A PA (1 +10 5 ) 1.4 ± 0.1 1.4 ± 0.4 0 ± 0 B PA (1 +10 7 ) 2.4 ± 0.8a 2.7 ± 0.7a 1.7 ± 1.4 C PA (1 +10 8 ) 1.7 ± 0.1 2.2 ± 1.0a 1.7 ± 0.4 3 A KP (1 +10 5 ) 1.7 ± 0.2 1.1 ± 0.6 1.0 ± 0.8 B KP (1 +10 7 ) 2.5 ± 0.6a 2.4 ± 1.5a 3.2 ± 1.2a C KP (1 +10 8 ) 3.1 ± 1.6a 3.2 ± 1.6a 3.4 ± 1.2a a Radiographic score was greater than 2. of bone matrix or marrow. Bone matrix and marrow suspensions were vortexed for 2 minutes and serially diluted with sterile physiological saline; 20 ml aliquots were plated onto blood agar, incubated at 37 C, and colonies counted after 24 hours. The limit of detection for viable counts was 50 colony-forming units per ml (CFUs/mL), corresponding to 150 CFUs/g of bone matrix and 200 CFUs/g of marrow. Means ± standard deviations were calculated and a twoway analysis of variance was used to determine whether there were significant differences in radiographical scoring or bacterial counts in matrix and marrow from left tibiae in rabbits from different groups. Differences between groups were deemed statistically significant if p 0.05. RESULTS Bacteria load and clearance rates for all groups are shown in Table I. Colony-forming units per gram of marrow and bone for the mono-organism groups show that AB, KP, or PA alone at 10 7 CFUs/mL was effective for rabbit osteomyelitis induction. Colony-forming units per gram of marrow and bone for the multibacterial groups show that the combination of AB (10 7 CFUs/mL)/KP (10 7 CFUs/mL)/PA (10 7 CFUs/mL)/MRSA (10 5 CFUs/mL) with 0.2 ml sodium morrhuate yielded a 100% osteomyelitis induction rate in our study. Radiographic results reflect the rate and extent of bone reconstruction and remodeling, which in osteomyelitis usually lags behind bacterial clearance. 13 Two weeks, radiographic scores for all mono-infection groups were varied (Table III). By 8 weeks, only one monoinfection group, PA (1 +10 5 CFUs/mL), showed significant radiographic improvement ( p < 0.05). For multibacterial osteomyelitis models, radiographic scores (Table IV) for six of the 10 groups were greater than Grade 2. By 8 weeks, however, only 1 group (KP [1 +10 7 CFUs/mL] + AB [1 +10 7 CFUs/mL] + PA [1 10 7 CFUs/mL] + MRSA [1 +10 5 CFUs/mL]) showed significant radiographic improvement ( p < 0.05). There were no adverse events. Of the 95 rabbits infected, none died before completion of treatment. All rabbits gained weight and tolerated the development of the severe tibial bone infection. DISCUSSION We proposed to adapt the existing rabbit model for the induction of osteomyelitis to simulate infection in trauma seen in U.S. military personnel from recent conflicts, particularly blast wounds to the extremities. This adaptation would offer the possibility of more accurately simulating the challenge of treating these wounds with available antibiotics or combinations of antibiotics. Our study has limitations typical of the established rabbit model. They are the possibility of self-recovery because of + TABLE IV. Radiographic Scores for the Multibacterial War Wound Groups Demonstrating That Only One Group (5A) Showed Significant Improvement at 8 Weeks Groups Inoculation Titer (CFUs/mL) 2 weeks 6 weeks 8 weeks 4 A AB (1 +10 8 ) + 2.0 ± 0.4a 0.9 ± 0.9 0.5 ± 0.4 B PA (1 +10 7 ) + 1.5 ± 0.6 1.0 ± 0.7 1.3 ± 0.7 C KP (1 +10 7 ) + 1.8 ± 0.7 2.5 ± 1.3a 1.0 ± 1.1 5 A AB (1 +10 7 ) + PA (1 +10 7 ) + 2.1 ± 0.4a 2.9 ± 1.6a 1.9 ± 0.8 B PA (1 +10 7 ) + KP (1 +10 7 ) + 1.8 ± 0.3 3.5 ± 0.7a 2.1 ± 0.8a C KP (1 +10 7 ) + AB (1 +10 7 ) + 2.2 ± 1.1a 1.9 ± 1.4 1.5 ± 0.6 D KP (1 +10 7 ) + AB (1 +10 7 ) + PA (1 +10 7 )2.4 ± 0.4a 1.5 ± 1.4 2.1 ± 1.1a 6 A KP (1x10 7 ) + AB (1 +10 7 ) + PA (1 +10 7 ) + 2.5 ± 0.9a 2.4 ± 1.2a 2.3 ± 1.3a B KP (1 +10 7 ) + AB (1 +10 7 ) + PA (1 +10 7 ) + 2.9 ± 0.6a 2.3 ± 1.5a 1.5 ± 0.9 C KP (1 +10 7 ) + AB (1 +10 7 ) + PA (1 +10 7 ) + 2.7 ± 1.4a 1.5 ± 1.22.7 ± 1.2a a Radiographic score was greater than 2. 698

animal immunity and infection to other sites or systemic sepsis because of the bacteremia. However, our subjects did not experience either problem. Although our study was sufficient in number of subjects to yield statistically significant results in radiographic scoring, we acknowledge that larger sample sizes will be needed to investigate further differences among treatment groups. Finally, our study did not use a true control or sham group of rabbits injected only with sodium morrhuate and no bacterial pathogen. In this regard, it should be noted that Scheman in 1941, using a 23-gauge needle and 0.3 to 0.4 ccs of sodium morrhuate, first showed that sodium morrhuate could produce either aseptic necrosis (when used alone) or osteomyelitis (when bacteria were also introduced) in the rabbit. 14 Norden, in 1970, further showed that animals injected with sodium morrhuate alone did not have pathologic evidence or cultures of osteomyelitis after 14 or 60 days, and that both bone necrosis (or the introduction of an implant, such as an intramedullary pin) and a bacterial pathogen were required to create an infection in the laboratory rabbit. 15 Although our model does not expose the rabbits to the level of trauma that is often seen in the field of battle, the use of sodium morrhuate simulates the necrosis that is often observed in war wounds. 16 Sodium morrhuate is a sclerosing agent that has been used in other rabbit studies to simulate damage to tissues such as cartilage and tendons. 17,18 It is also used in various animal models of infection, 19 23 including traumatic infection, 24 although researchers who are not studying trauma typically may prefer to use devascularized bone 25 or more likely an implant 26 to reliably produce the infections required to test antibiotics in animals. Although our study does not use an implant, the presence of surrounding tissue necrosis in war wounds complicates treatment for infection, and this is compounded further when multiple strains of bacteria are present. The rabbit osteomyelitis models developed by previous researchers were designed for a monobacterial approach, which can be treated by single or combined antibiotic therapy. 1,2,15,26 30 We have shown a method to broaden the model to study multibacterial infections. The data show that AB, KP, or PA alone at 10 7 CFUs/mL was effective for rabbit osteomyelitis induction. The different combinations of MDROs were more effective in osteomyelitis induction. The combination of AB (10 7 CFUs/mL)/KP (10 7 CFUs/mL)/PA (10 7 CFUs/mL)/MRSA (10 5 CFUs/mL) with 0.2 ml sodium morrhuate yielded 100% osteomyelitis induction rate in our pilot study. The radiographic images showed all stages of osteomyelitis as that of human infection. All the rabbits gained weight and tolerated the severe tibial infection by multiple drug resistant organisms. Our results suggest that AB, PA, or KP alone, or in combinations including MRSA was effective in the development of MDRO-induced war wound rabbit osteomyelitis. Such an animal model could be reliably reproducible in large numbers. We have initiated larger animal trials to examine the efficacy of single antibiotics or antibiotic combinations, including tigecycline, daptomycin, and vancomycin, to determine which treatment regimen is most effective in eradicating MDRO infections in this model. ACKNOWLEDGMENTS The authors acknowledge Clinton K. Murray, MD, Brooke Army Med Center, Fort Sam Houston, Texas, for assistance in obtaining bacterial samples, and Stephen McConoughey, PhD, and Randy Roberts of The Ohio State University for their assistance in preparing this manuscript. 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