Janne Koch

A porcine model of acute, haematogenous, localized osteomyelitis due to Staphylococcus aureus: a pathomorphological study

Johansen LK, Frees D, Aalbæk B, Koch J, Iburg T, Nielsen OL, Leifsson PS, Jensen HE. A porcine model of acute, haematogenous, localized osteomyelitis due to Staphylococcus aureus: a pathomorphological study, APMIS 2010; 119: 111–8.

Pathology and biofilm formation in a porcine model of staphylococcal osteomyelitis.

A porcine model was used to examine the potential of human and porcine Staphylococcus aureus isolates to induce haematogenously spread osteomyelitis. Pigs were inoculated in the right femoral artery with one of the following S. aureus strains: S54F9 (from a porcine lung abscess; n = 3 animals), NCTC-8325-4 (a laboratory strain of human origin; n = 3 animals) and UAMS-1 (a human osteomyelitis isolate; n = 3 animals). Two pigs were sham inoculated with saline. At 11 or 15 days post infection the animals were scanned by computed tomography before being killed and subjected to necropsy examination. Osteomyelitis lesions were present in the right hind limb of all pigs inoculated with strain S54F9 and in one pig inoculated with strain NCTC-8325-4. Microscopically, there was extensive loss of bone tissue with surrounding granulation tissue. Sequestrated bone trabeculae were intermingled with colonies of S. aureus as demonstrated immunohistochemically. By peptide nucleic acid fluorescence in situ hybridization bacterial aggregates were demonstrated to be embedded in an opaque matrix, indicating that the bacteria had formed a biofilm. Development of experimental osteomyelitis was therefore dependent on the strain of bacteria inoculated and on the formation of a biofilm.

A New Technique for Modeling of Hematogenous Osteomyelitis in Pigs: Inoculation into Femoral Artery

ABSTRACT A new inoculation technique has been developed and applied in a porcine model of juvenile hematogenous osteomyelitis. Following the success of the model, we describe the inoculation technique in detail to enable its replication in future studies. The technique was based on an anatomical feature of the femoral artery that enables inoculation into the artery using a simple surgical procedure. Inoculation in the femoral artery is advantageous because the localization of lesions constitutes a discriminative model of the naturally occurring hematogenous osteomyelitis in long bones, usually involving femur and tibia in children. The procedure was performed under general anesthesia and consisted of five major steps: (1) Exposure of the right femoral artery, (2) retrograde catheterization, (3) inoculation of bacteria, (4) hemostasis of the arterial puncture site using compression, and (5) suturing of the wound in two layers.

Effects of Implant-associated Osteomyelitis on Cefuroxime Bone Pharmacokinetics: Assessment in a Porcine Model

BACKGROUND The prolonged antibiotic therapy that is often needed for successful management of osteomyelitis may be related to incomplete penetration of antibiotics into the target site. The objective of this study was to assess the effects of implant-associated osteomyelitis on cefuroxime penetration into bone. METHODS Implant-associated osteomyelitis using a Staphylococcus aureus strain was induced in the right tibia in ten pigs. After five days and following administration of 1500 mg of cefuroxime, measurements of cefuroxime were obtained using microdialysis for eight hours in the implant-related bone cavity, in the adjacent infected cancellous bone and infected subcutaneous tissue, and in healthy cancellous bone and subcutaneous tissue in the contralateral leg. Measurements of the corresponding free plasma concentrations were also obtained. The extent of the infection was assessed by postmortem computed tomography (CT) scans and cultures of blood, swabs, and bone specimens. RESULTS Bone destruction was found in the implant cavities. No structural bone changes in the adjacent infected cancellous bone were visible on CT scans. S. aureus was grown on culture of specimens from all implant cavities and from eight of ten swabs and seven of ten bone samples from the infected bone. The areas under the concentration-time curves for the different tissues differed significantly, with the lowest area under the curve found in the implant cavity (analysis of variance; p < 0.001). Although not as notable as for the implant cavity, cefuroxime penetration into infected cancellous bone was incomplete but comparable with that in healthy bone. Despite poorer tissue penetration, slightly increased time with concentrations above the minimal inhibitory concentration (MIC) was achieved in the implant cavity up to MICs of 2 mg/L compared with the other tissues, but the time was shorter for higher MICs. CONCLUSIONS Cefuroxime penetration into infected cancellous bone was incomplete but comparable with that in healthy bone. The destructive bone processes associated with acute osteomyelitis reduced cefuroxime penetration further. CLINICAL RELEVANCE These findings support the general clinical perception that fast diagnosis and early initiation of antibiotics before the development of implant-associated cavities is important in nonsurgical management of acute osteomyelitis.

Systemic inflammatory response and local cytokine expression in porcine models of endocarditis

The knowledge of systemic inflammation and local cytokine expression in porcine endocarditis models is limited, though it could provide valuable information about the pathogenesis and comparability to human endocarditis. Analyses of bacteriology and hematology were performed on blood samples from pigs with non‐bacterial thrombotic endocarditis (NBTE, n = 11), Staphylococcus aureus infective endocarditis (IE, n = 2), animals with S. aureus sepsis without endocarditis (n = 2) and controls (n = 2). Furthermore, immunohistochemistry was used to examine the local expression of IL‐1β and IL‐8. Bacterial blood cultures were continuously positive in IE pigs from inoculation to euthanasia, and negative in all other pigs at all times. The total white blood cell counts and total neutrophil counts were massively elevated in pigs with IE. Local IL‐1β and IL‐8 expression in IE pigs were moderate to high, and high, respectively. In addition, slight local expression of IL‐1β and IL‐8 was present in some NBTE pigs. In the IE model, both the systemic inflammatory response and the high local expression of IL‐8 were comparable to the human disease. Furthermore, the results indicate IL‐1β and IL‐8 as important contributors in the endocarditis pathogenesis.

A novel knee prosthesis model of implant-related osteo- myelitis in rats

Background and purpose There have been numerous reports of animal models of osteomyelitis. Very few of these have been prosthesis models that imitate human conditions. We have developed a new rat model of implant-related osteomyelitis that mimics human osteomyelitis, to investigate the pathology of infection after orthop edic implant surgery. Methods 2 wild-type strains of Staphylococcus aureus, MN8 and UAMS-1, and their corresponding mutants that are unable to produce poly-N-acetyl glucosamine (PNAG) (ica::tet) were injected into the medullary canals of the femur and tibia at 3 different doses: 102, 103, and > 104 CFU/rat. We measured clinical signs, inflammatory markers, radiographic signs, histopathology, and bacteriology in the infected animals. Results An inoculum of at least 104 cfu of either wild-type bacterial strain resulted in histological, bacteriological, and radiographic signs of osteomyelitis with loosening of the prosthesis. An inoculum of 103 CFU gave signs of osteomyelitis but the prosthesis remained in situ. Bacterial inocula of 102 cfu gave no signs of osteolysis. Interpretation We have established a new knee prosthesis model that is suitable for reliable induction of experimental implant-associated osteomyelitis with the prosthesis in situ, using a small inoculum of S. aureus. At a dose of 103 CFU/rat, bacteria unable to produce PNAG (ica::tet) had only minor defects in their virulence.

A novel porcine model of implant associated osteomyelitis: a comprehensive analysis of local, regional and systemic response

Pigs are favorable experimental animals for infectious diseases in humans. However, implant‐associated osteomyelitis (IAO) models in pigs have only been evaluated using high‐inoculum infection (>108 CFU) models in 1975 and 1993. Therefore, the aim of this paper was to present a new low inoculum porcine model of human IAO based on 42 experimental pigs. The model was created by drilling an implant cavity in the tibial bone followed by insertion of a small steel implant and simultaneous inoculation of Staphylococcus aureus bacteria (n = 32) or saline (n = 10). The infected pigs were either inoculated with 104 CFU (n = 26) or 102 and 103 CFU (n = 6). All animals were euthanized 5 days after insertion of implants. Pigs receiving the high‐inoculum infections showed a significantly higher volume of bone lesion, number of neutrophils around the implant, concentrations of acute phase proteins in serum, and enlargement of regional lymph nodes. A positive correlation was present between a high number of surrounding neutrophils and high values of all other parameters. Furthermore, a threshold of 40 neutrophils per 10 high power fields for the histopathological diagnosis of high grade IAO was defined. In conclusion: This paper describes a novel low‐inoculum S. aureus porcine model of IAO which was demonstrated to be reliable, reproducible and discriminative to human IAO, and represents a requested and valuable tool in orthopedic research.

Immune Cells from SR/CR Mice Induce the Regression of Established Tumors in BALB/c and C57BL/6 Mice

Few experimental models are available for the study of natural resistance to cancer. One of them is the SR/CR (spontaneous regression/complete resistance) mouse model in which natural resistance to a variety of cancer types appeared to be inherited in SR/CR strains of BALB/c and C57BL/6 mice. The genetic, cellular, and molecular effector mechanisms in this model are largely unknown, but cells from the innate immune system may play a significant role. In contrast to previous observations, the cancer resistance was limited to S180 sarcoma cancer cells. We were unable to confirm previous observations of resistance to EL-4 lymphoma cells and J774A.1 monocyte-macrophage cancer cells. The cancer resistance against S180 sarcoma cells could be transferred to susceptible non-resistant BALB/c mice as well as C57BL/6 mice after depletion of both CD4+/CD8+ leukocytes and B-cells from SR/CR mice. In the responding recipient mice, the cancer disappeared gradually following infiltration of a large number of polymorphonuclear granulocytes and remarkably few lymphocytes in the remaining tumor tissues. This study confirmed that the in vivo growth and spread of cancer cells depend on a complex interplay between the cancer cells and the host organism. Here, hereditary components of the immune system, most likely the innate part, played a crucial role in this interplay and lead to resistance to a single experimental cancer type. The fact that leukocytes depleted of both CD4+/CD8+ and B cells from the cancer resistant donor mice could be transferred to inhibit S180 cancer cell growth in susceptible recipient mice support the vision of an efficient and adverse event free immunotherapy in future selected cancer types.

Early implant-associated osteomyelitis results in a peri-implanted bacterial reservoir

Implant‐associated osteomyelitis (IAO) is a common complication in orthopedic surgery. The aim of this study was to elucidate how deep IAO can go into the peri‐implanted bone tissue within a week. The study was performed in a porcine model of IAO. A small steel implant and either 104 CFU/kg body weight of Staphylococcus aureus or saline was inserted into the right tibial bone of 12 pigs. The animals were consecutively killed on day 2, 4 and 6 following implantation. Bone tissue around the implant was histologically evaluated. Identification of S. aureus was performed immunohistochemically on tissue section and with scanning electron microscopy and peptide nucleic acid in situ hybridization on implants. The distance of the peri‐implanted pathological bone area (PIBA), measured perpendicular to the implant, was significantly larger in infected animals compared to controls (p = 0.0014). The largest differences were seen after 4 and 6 days of inoculation, where PIBA measurements of up to 6 mm were observed. Positive S. aureus bacteria were identified on implants and from 25 μm to 6 mm into PIBA. This is important knowledge for optimizing outcomes of surgical debridement in osteomyelitis.

Local osteogenic expression of cyclooxygenase-2 and systemic response in porcine models of osteomyelitis.

It is suggested that cyclooxygenase 2 (COX-2) derived prostaglandins contributes to the progressive bone loss seen in osteomyelitis lesions. In the present study we examined the expression of COX-2 in bones from 23 pigs with experimental osteomyelitis. Osteomyelitis was induced with Staphylococcus aureus and groups of animals were euthanized following 6 h, 12 h, 24 h, 2 days, 5 days, 11 days and 15 days, respectively. Expression of COX-2 was evaluated immunohistochemically and combined with characterization of morphological changes in bone tissue. Furthermore, the serum concentrations of alkaline phosphatase and haptoglobin were measured. Extensive COX-2 expression by osteoblasts was present 2 days after inoculation together with many activated osteoclasts. Simultaneously, the serum concentration of alkaline phosphatase decreased whereas the haptoglobin concentration increased. This is the first in vivo study showing an early wave of COX-2 mediated bone resorption during osteomyelitis. Therefore, treatment aiming to reduce the break down of bone tissue directed by the COX-2 pathway might be suggested early in the course of the disease.