Quantitative mouse model of implant-associated osteomyelitis and the kinetics of microbial growth, osteolysis, and humoral immunity

The Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Box 665, Rochester, New York 14642, USA.
Journal of Orthopaedic Research (Impact Factor: 2.99). 01/2008; 26(1):96-105. DOI: 10.1002/jor.20452
Source: PubMed


Although osteomyelitis (OM) remains a serious problem in orthopedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity, and osteolysis. To overcome these obstacles, we developed a murine model of implant-associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X-ray and micro-CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant-associated OM, namely: osteolysis, sequestrum formation, and involucrum of Gram-positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after 1 week, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 postinfection. Real-time quantitative PCR (RTQ-PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days postinfection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA-E transformed S. aureus (Xen29) combined with nuc RTQ-PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p < 0.05). Collectively, these studies demonstrate the first quantitative model of implant-associated OM that defines the kinetics of microbial growth, osteolysis, and humoral immunity following infection.

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Available from: Kjeld Søballe, Sep 15, 2014
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    • "While the majority of our current knowledge on biofilms stem from in vitro studies, animal models are in high demand to investigate the clinical aspects of biofilm infections. A murine model has been proposed using bioluminescence imaging (BLI) and quantitative PCR as quantitative endpoints for the bacterial load [12]. This model employs a PCR based detection of bacterial load from infected bone, but does not address the vital issue of biofilm formation on the implant. "
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    ABSTRACT: Bacterial biofilms causing implant-associated osteomyelitis is a severe complication with limited antimicrobial therapy options. We designed an animal model, in which implant associated osteomyelitis arise from a Staphylococcus aureus biofilm on a tibia implant. Two bioluminescently engineered (luxA-E transformed), strains of S. aureus were utilized, Xen29 and Xen31. Biofilm formation was assessed with epifluorescence microscopy. Quantitative measurements were performed day 4, 6, 8, 11 and 15 post-surgery. Bacteria were extracted from the biofilm by sonication and the bacterial load quantified by culturing. Biofilm formation was evident from day 6 post-implantation. Mean bacterial load from implants was ∼1×104 CFU/implant, while mean bacterial load from infected tibias were 1×106 CFU/bone. Bioluminesence imaging revealed decreasing activity throughout the 15-day observation period, with signal intensity for both strains reaching that of the negative control by day 15 while there was no significant reduction in bacterial load. The model is suitable for testing antimicrobial treatment options for implant associated OM, as treatment efficacy on both biofilm and viable counts can be assessed.
    Full-text · Article · Oct 2014 · PLoS ONE
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    • "Newly developed optical imaging techniques such as BLI and FRI enable the longitudinal real-time monitoring of bacterial growth in living animals, throughout the course of infection in various organs [2]–[6], [19], [23]. Models of bone or joint infection are commonly used to study musculoskeletal infections [2], [5], [21], [25]. A few animal models have been reported for soft tissue and skin infection, including the flank [4], [18], thigh [1], [8], and subcutaneous areas [9]. "
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    ABSTRACT: Musculoskeletal infections, including surgical-site and implant-associated infections, often cause progressive inflammation and destroy areas of the soft tissue. Treating infections, especially those caused by multi-antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) remains a challenge. Although there are a few animal models that enable the quantitative evaluation of infection in soft tissues, these models are not always reproducible or sustainable. Here, we successfully established a real-time, in vivo, quantitative mouse model of soft-tissue infection in the superficial gluteus muscle (SGM) using bioluminescence imaging. A bioluminescent strain of MRSA was inoculated into the SGM of BALB/c adult male mice, followed by sequential measurement of bacterial photon intensity and serological and histological analyses of the mice. The mean photon intensity in the mice peaked immediately after inoculation and remained stable until day 28. The serum levels of interleukin-6, interleukin-1 and C-reactive protein at 12 hours after inoculation were significantly higher than those prior to inoculation, and the C-reactive protein remained significantly elevated until day 21. Histological analyses showed marked neutrophil infiltration and abscesses containing necrotic and fibrous tissues in the SGM. With this SGM mouse model, we successfully visualized and quantified stable bacterial growth over an extended period of time with bioluminescence imaging, which allowed us to monitor the process of infection without euthanizing the experimental animals. This model is applicable to in vivo evaluations of the long-term efficacy of novel antibiotics or antibacterial implants.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "Mice were anesthetized via IP injection of 100 mg/kg Ketamine (Ketaset®; Fort Dodge Laboratories, Inc., Fort Dodge, Iowa) and 10 mg/kg Xylazine (Rugby Laboratories, Inc., Rockville Center, NY). The left leg of each mouse was cleansed with povidone iodine and rinsed with 70% ethanol before surgical implantation of an autoclaved 0.25 mm insect pin into the tibia (Fine Science Tools, Foster City, CA) as described [43], [44]. The implanted pin was then inoculated with 1 µl of the S. aureus SAP149 suspension. "
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    ABSTRACT: Staphylococcus aureus is a major human pathogen and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. While S. aureus protective antigens have been identified in the literature, the majority have only been tested in a single animal model of disease. We wished to evaluate the ability of one S. aureus vaccine antigen to protect in multiple mouse models, thus assessing whether protection in one model translates to protection in other models encompassing the full breadth of infections the pathogen can cause. We chose to focus on genetically inactivated alpha toxin mutant HlaH35L. We evaluated the protection afforded by this antigen in three models of infection using the same vaccine dose, regimen, route of immunization, adjuvant, and challenge strain. When mice were immunized with HlaH35L and challenged via a skin and soft tissue infection model, HlaH35L immunization led to a less severe infection and decreased S. aureus levels at the challenge site when compared to controls. Challenge of HlaH35L-immunized mice using a systemic infection model resulted in a limited, but statistically significant decrease in bacterial colonization as compared to that observed with control mice. In contrast, in a prosthetic implant model of chronic biofilm infection, there was no significant difference in bacterial levels when compared to controls. These results demonstrate that vaccines may confer protection against one form of S. aureus disease without conferring protection against other disease presentations and thus underscore a significant challenge in S. aureus vaccine development.
    Full-text · Article · Apr 2013 · PLoS ONE
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