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Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections. In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden. To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia. While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

Preclinical in vitro and in vivo methods to study bacterial interactions with dermal fillers and infection pathogenesis are lacking. In this work, first in vitro methods to assess protein biofouling and effective pore size of commercial dermal fillers, including degradable hyaluronic acid (HA)-based fillers and other semi-degradable or permanent fillers (non-HA), were developed. The results were then related to Staphylococcus aureus (S. aureus) adhesion rates in vitro. HA fillers had less protein sorption than non-HA fillers and overall had smaller effective pore sizes. The properties correlated with levels of bacterial adhesion, where the control glass surface had the most rapid increase in bacterial cell adhesion, with a slope of 0.29 cm-2  min-1 , three unique non-HA fillers had intermediate adhesion with slopes of 0.11 and 0.06 cm-2  min-1 , and three unique HA fillers had the least adhesion with slopes of 0.02, 0.02, and 0.01 cm-2  min-1 . S. aureus had greater motility on the HA fillers than on non-HA fillers. Next, a mouse model for dermal filler biofilm and infection was developed. Mice were inoculated with a controlled amount of bioluminescent bacteria (Xen36 S. aureus) and polyacrylamide hydrogels of different stiffness were injected. In vivo bioluminescence was monitored longitudinally for 35 days to ensure that lasting colonization was established. The inoculum was optimized to achieve adequate bioluminescent signal, and bacterial bioburden over time and inter-animal variability in bioburden were determined. These in vitro and in vivo approaches can be used for future studies of antimicrobial interventions for dermal fillers.

Implant-associated infections are challenging to diagnose and treat. Fluorescent probes have been heralded as a technologic advancement that can improve our ability to non-invasively identify infecting organisms, as well as guide the inexact procedure of surgical debridement. This study's purpose was to compare two fluorescent probes for their ability to localize Staphylococcus aureus biofilm infections on spinal implants utilizing noninvasive optical imaging, then assessing the broader applicability of the more successful probe in other infection animal models. This was followed by real-time, fluorescence image-guided surgery to facilitate debridement of infected tissue. The two probe candidates, a labelled antibiotic that targets peptidoglycan (Vanco-800CW), and the other, a labelled antibody targeting the immunodominant Staphylococcal antigen A (1D9-680), were injected into mice with spine implant infections. Mice were then imaged noninvasively with near infrared fluorescent imaging at wavelengths corresponding to the two probe candidates. Both probes localized to the infection, with the 1D9-680 probe showing greater fidelity over time. The 1D9-680 probe was then tested in mouse models of shoulder implant and allograft infection, demonstrating its broader applicability. Finally, an image-guided surgery system which superimposes fluorescent signals over analog, real-time, tissue images was employed to facilitate debridement of fluorescent-labelled bacteria.

BackgroundAnimal models are used to guide management of periprosthetic implant infections. No adequate model exists for periprosthetic shoulder infections, and clinicians thus have no preclinical tools to assess potential therapeutics. We hypothesize that it is possible to establish a mouse model of shoulder implant infection (SII) that allows noninvasive, longitudinal tracking of biofilm and host response through in vivo optical imaging. The model may then be employed to validate a targeting probe (1D9-680) with clinical translation potential for diagnosing infection and image-guided débridement.MethodsA surgical implant was press-fit into the proximal humerus of c57BL/6J mice and inoculated with 2 μL of 1 × 103 (e3), or 1 × 104 (e4), colony-forming units (CFUs) of bioluminescent Staphylococcus aureus Xen-36. The control group received 2 μL sterile saline. Bacterial activity was monitored in vivo over 42 days, directly (bioluminescence) and indirectly (targeting probe). Weekly radiographs assessed implant loosening. CFU harvests, confocal microscopy, and histology were performed.ResultsBoth inoculated groups established chronic infections. CFUs on postoperative day (POD) 42 were increased in the infected groups compared with the sterile group (P < .001). By POD 14, osteolysis was visualized in both infected groups. The e4 group developed catastrophic bone destruction by POD 42. The e3 group maintained a congruent shoulder joint. Targeting probes helped to visualize low-grade infections via fluorescence.DiscussionGiven bone destruction in the e4 group, a longitudinal, noninvasive mouse model of SII and chronic osteolysis was produced using e3 of S aureus Xen-36, mimicking clinical presentations of chronic SII.ConclusionThe development of this model provides a foundation to study new therapeutics, interventions, and host modifications.

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections. In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden. To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia. While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. However, the relatively short emission wavelength of lux (peak 490 nm) has limited tissue penetration. To overcome this limitation, the gene for the click beetle (Pyrophorus plagiophtalamus) red luciferase (luc) (with a longer >600 emission wavelength), was introduced singly and in combination with the lux operon into a methicillin-resistant S. aureus strain. After administration of the substrate D-luciferin, the luc bioluminescent signal was substantially greater than the lux signal in vitro. The luc signal had enhanced tissue penetration and improved anatomical co-registration with infected internal organs compared with the lux signal in a mouse model of S. aureus bacteremia with a sensitivity of approximately 3 × 104 CFU from the kidneys. Finally, in an in vivo mixed bacterial wound infection mouse model, S. aureus luc signals could be spectrally unmixed from Pseudomonas aeruginosa lux signals to noninvasively monitor the bacterial burden of both strains. Therefore, the S. aureus luc reporter may provide a technological advance for monitoring invasive organ dissemination during S. aureus bacteremia and for studying bacterial dynamics during mixed infections.

Spine implant infections portend disastrous outcomes, as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. Current imaging modalities can detect anatomical alterations and anomalies but cannot differentiate between infection and aseptic loosening, diagnose specific pathogens, or delineate the extent of an infection. Herein, a fully human monoclonal antibody 1D9, recognizing the immunodominant staphylococcal antigen A on the surface of Staphylococcus aureus, was assessed as a nuclear and fluorescent imaging probe in a preclinical model of S. aureus spinal implant infection, utilizing bioluminescently labeled bacteria to confirm the specificity and sensitivity of this targeting. Postoperative mice were administered 1D9 probe dual labeled with 89-zirconium (89Zr) and a near infrared dye (NIR680) (89Zr-NIR680-1D9), and PET-CT and in vivo fluorescence and bioluminescence imaging were performed. The 89Zr-NIR680-1D9 probe accurately diagnosed both acute and subacute implant infection and permitted fluorescent image-guided surgery for selective debridement of infected tissue. Therefore, a single probe could noninvasively diagnose an infection and facilitate image-guided surgery to improve the clinical management of implant infections.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

[This corrects the article DOI: 10.3389/fmicb.2019.00159.].

Short-chain fatty acids (SCFA) are important dietary and microbiome metabolites that can have roles in gut immunity as well as further afield. We previously observed that gut microbiome alteration via antibiotics led to attenuated lung inflammatory responses. The rationale for this study was to identify gut microbiome factors that regulate lung immune homeostasis. We first investigated key factors within mouse colonic lumen filtrates (CLF) which could elicit direct inflammatory effects in vitro. We identified lipopolysaccharide (LPS) and SCFAs as key CLF ingredients whose levels and inflammatory capacity changed after antibiotic exposure in mice. Specifically, the SCFA propionate appeared to be a key regulator of LPS responses in vitro. Elevated propionate: acetate ratios, as seen in CLF after antibiotic exposure, strongly blunted inflammatory responses in vitro. In vivo, exposure of lungs to high dose propionate, to mimic how prior antibiotic exposure changed SCFA levels, resulted in diminished immune containment of Staphylococcus aureus pneumonia. Finally, we discovered an enrichment of propionate-producing gut bacteria in mice with reduced lung inflammation following lung ischemia reperfusion injury in vivo. Overall, our data show that propionate levels can distinctly modulate lung immune responses in vitro and in vivo and that gut microbiome increased production of propionate is associated with reduced lung inflammation.

Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, 89Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.

Bacterial biofilm infections of implantable medical devices decrease the effectiveness of antibiotics, creating difficult-to-treat chronic infections. Prosthetic joint infections (PJI) are particularly problematic because they require prolonged antibiotic courses and reoperations to remove and replace the infected prostheses. Current models to study PJI focus on Gram-positive bacteria, but Gram-negative PJI (GN-PJI) are increasingly common and are often more difficult to treat, with worse clinical outcomes. Herein, we sought to develop a mouse model of GN-PJI to investigate the pathogenesis of these infections and identify potential therapeutic targets. An orthopedic-grade titanium implant was surgically placed in the femurs of mice, followed by infection of the knee joint with Pseudomonas aeruginosa or Escherichia coli. We found that in vitro biofilm-producing activity was associated with the development of an in vivo orthopedic implant infection characterized by bacterial infection of the bone/joint tissue, biofilm formation on the implants, reactive bone changes, and inflammatory immune cell infiltrates. In addition, a bispecific antibody targeting P. aeruginosa virulence factors (PcrV and Psl exopolysaccharide) reduced the bacterial burden in vivo. Taken together, our findings provide a preclinical model of GN-PJI and suggest the therapeutic potential of targeting biofilm-associated antigens.

Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, 89Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.

IntroductionDiagnosing prosthetic joint infection (PJI) poses significant challenges, and current modalities are fraught with low sensitivity and/or potential morbidity. Photoacoustic imaging (PAI) is a novel ultrasound-based modality with potential for diagnosing PJI safely and noninvasively.MaterialsIn an established preclinical mouse model of bioluminescent Staphylococcus aureus PJI, fluorescent indocyanine green (ICG) was conjugated to β-cyclodextrin (CDX-ICG) or teicoplanin (Teic-ICG) and injected intravenously for 1 week postoperatively. Daily fluorescent imaging and PAI were used to localize and quantify tracer signals. Results were analyzed using 2-way analysis of variance.ResultsFluorescence clearly localized to the site of infection and was significantly higher with Teic-ICG compared with CDX-ICG (P = 0.046) and ICG alone (P = 0.0087). With PAI, the photoacoustic signal per volumetric analysis was substantially higher and better visualized with Teic-ICG compared with CDX-ICG and ICG alone, and colocalized well with bioluminescence and fluorescence imaging.ConclusionPhotoacoustic imaging successfully localized PJI in this proof-of-concept study and demonstrates potential for clinical translation in orthopaedics.

Post-operative spine infections are a challenge, as hardware must often be retained to prevent destabilization of the spine, and bacteria form biofilm on implants, rendering them inaccessible to antibiotic therapy, and immune cells. A model of posterior-approach spinal surgery was created in which a stainless steel k-wire was transfixed into the L4 spinous process of 12-week-old C57BL/six mice. Mice were then randomized to receive either one of three concentrations (1 × 102 , 1 × 103 , and 1 × 104 colony forming units (CFU)) of a bioluminescent strain of Staphylococcus aureus or normal saline at surgery. The mice were then longitudinally imaged for bacterial bioluminescence to quantify infection. The 1 × 102 CFU group had a decrease in signal down to control levels by POD 25, while the 1 × 103 and 1 × 104 CFU groups maintained a 10-fold higher signal through POD 35. Bacteria were then harvested from the pin and surrounding tissue for confirmatory CFU counts. All mice in the 1 × 104 CFU group experienced wound breakdown, while no mice in the other groups had this complication. Once an optimal bacterial concentration was determined, mice expressing enhanced green fluorescent protein in their myeloid cells (Lys-EGFP) were utilized to contemporaneously quantify bacterial burden, and immune response. Neutrophil fluorescence peaked for both groups on POD 3, and then declined. The infected group continued to have a response above the control group through POD 35. This study, establishes a noninvasive in vivo mouse model of spine implant infection that can quantify bacterial burden and host inflammation longitudinally in real time without requiring animal sacrifice. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:193-199, 2017.

BackgroundManagement of spine implant infections (SII) are challenging. Explantation of infected spinal hardware can destabilize the spine, but retention can lead to cord compromise and biofilm formation, complicating management. While vancomycin monotherapy is commonly used, in vitro studies have shown reduced efficacy against biofilm compared to combination therapy with rifampin. Using an established in vivo mouse model of SII, we aim to evaluate whether combination therapy has increased efficacy compared to both vancomycin alone and infected controls.MethodsAn L-shaped, Kirschner-wire was transfixed into the L4 spinous process of 12-week-old C57BL/6 mice, and inoculated with bioluminescent Staphylococcus aureus. Mice were randomized into a vancomycin group, a combination group with vancomycin plus rifampin, or a control group receiving saline. Treatment began on post-operative day (POD) 7 and continued through POD 14. In vivo imaging was performed to monitor bioluminescence for 35 days. Colony-forming units (CFUs) were cultured on POD 35.ResultsBioluminescence peaked around POD 7 for all groups. The combination group had a 10-fold decrease in signal by POD 10. The vancomycin and control groups reached similar levels on POD 17 and 21, respectively. On POD 25 the combination group dropped below baseline, but rebounded to the same level as the other groups, demonstrating a biofilm-associated infection by POD 35. Quantification of CFUs on POD 35 confirmed an ongoing infection in all three groups.ConclusionsAlthough both therapies were initially effective, they were not able to eliminate implant biofilm bacteria, resulting in a rebound infection after antibiotic cessation. This model shows, for the first time, why histologic-based, static assessments of antimicrobials can be misleading, and the importance of longitudinal tracking of infection. Future studies can use this model to test combinations of antibiotic therapies to see if they are more effective in eliminating biofilm prior to human trials.

Sterile lung injury is an important clinical problem that complicates the course of severely ill patients. Interruption of blood flow, namely ischemia-reperfusion (IR), initiates a sterile inflammatory response in the lung that is believed to be maladaptive. The rationale for this study was to elucidate the molecular basis for lung IR inflammation and whether it is maladaptive or beneficial. Using a mouse model of lung IR, we demonstrate that sequential blocking of inflammasomes [specifically, NOD-, LRR-, and pyrin domain-containing 3 (NLRP3)], inflammatory caspases, and interleukin (IL)-1β, all resulted in an attenuated inflammatory response. IL-1β production appeared to predominantly originate in conjunction with alveolar type 2 epithelial cells. Lung IR injury recruited unactivated or dormant neutrophils producing less reactive oxygen species thereby challenging the notion that recruited neutrophils are terminally activated. However, lung IR inflammation was able to limit or reduce the bacterial burden from subsequent experimentally induced pneumonia. Notably, inflammasome-deficient mice were unable to alter this bacterial burden following IR. Thus, we conclude that the NLRP3 inflammasome, through IL-1β production, regulates lung IR inflammation, which includes recruitment of dormant neutrophils. The sterile IR inflammatory response appears to serve an important function in inducing resistance to subsequent bacterial pneumonia and may constitute a critical part of early host responses to infection in trauma.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and μCT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, μCT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical μCT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with μCT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and μCT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, μCT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical μCT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with μCT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

The incidence of infections related to cardiac devices (such as permanent pacemakers) has been increasing out of proportion to implantation rates. As management of device infections typically requires explantation of the device, optimal prophylactic strategies are needed. Cefazolin and vancomycin are widely used as single agents for surgical prophylaxis against cardiac device-related infections. However, combination antibiotic prophylaxis may further reduce infectious complications. To model a localized subcutaneous implant-related infection, a bioluminescent strain of Staphylococcus epidermidis was inoculated onto a medical-procedure-grade titanium disc, which was placed into a subcutaneous pocket in the backs of mice. In vivo bioluminescence imaging, quantification of ex vivo CFU from the capsules and implants, variable-pressure scanning electron microscopy (VP-SEM), and neutrophil enhanced green fluorescent protein (EGFP) fluorescence in LysEGFP mice were employed to monitor the infection. This model was used to evaluate the efficacies of low- and high-dose cefazolin (50 and 200 mg/kg of body weight) and vancomycin (10 and 110 mg/kg) intravenous prophylaxis with or without rifampin (25 mg/kg). High-dose cefazolin and high-dose vancomycin treatment resulted in almost complete bacterial clearance, whereas both low-dose cefazolin and low-dose vancomycin reduced the in vivo and ex vivo bacterial burden only moderately. The addition of rifampin to low-dose cefazolin and vancomycin was highly effective in further reducing the CFU harvested from the implants. However, vancomycin-rifampin was more effective than cefazolin-rifampin in further reducing the CFU harvested from the surrounding tissue capsules. Future studies in humans will be required to determine whether the addition of rifampin has improved efficacy in preventing device-related infections in clinical practice.

IntroductionPhysical activity reduces fatigue and depression while improving quality of life in cancer survivors. Exercise is generally considered safe and is recommended to survivors of all ages. Despite the high prevalence of cancer in the elderly, few studies address physical activity interventions targeting this older population. Fit & Strong! is an evidence-based physical activity program shown to improve level of physical activity, exercise-self-efficacy, and mood in older adults with osteoarthritis. This study tests the feasibility and short-term impact of the Fit & Strong! exercise program adapted for older cancer survivors.MethodsParticipants were cancer survivors at least 50 years of age who were not on active treatment with intravenous chemotherapy or radiation. They participated in the 8-week Fit & Strong! program, which included three 90-min sessions per week; 60 min of group physical activity and 30 min of education. Education on osteoarthritis was removed from the Fit & Strong! program and replaced with relevant topics on cancer survivorship issues. Feasibility was measured by the ability to recruit and retain older cancer survivors. Pre and post-intervention surveys evaluated the effect of the intervention on physical activity and quality of life.ResultsThe study enrolled 72 cancer survivors to participate in an 8-week exercise program. The mean age of participants was 70. Over two-thirds (68%) of participants completed the program and with a mean attendance rate of 75% (18 of 24 sessions). No safety issues occurred. Improvements from baseline to post-intervention were observed for self-reported minutes of physical activity per week, self-efficacy for aerobic exercise, and symptoms related to depression and anxiety.ConclusionThis study was successful in recruiting and retaining a population of older cancer survivors to participate in a group exercise program. Significant improvement in level of physical activity and mood suggests this evidence-based physical activity intervention can be adapted to promote health benefits in cancer survivors. Additional studies are necessary to confirm efficacy and assess long-term benefits.

The incidence of infections related to cardiac devices (such as permanent pacemakers) has been increasing out of proportion to implantation rates. As management of device infections typically requires explantation of the device, optimal prophylactic strategies are needed. Cefazolin and vancomycin are widely used as single agents for surgical prophylaxis against cardiac device-related infections. However, combination antibiotic prophylaxis may further reduce infectious complications. To model a localized subcutaneous implant-related infection, a bioluminescent strain of Staphylococcus epidermidis was inoculated onto a medical-procedure-grade titanium disc, which was placed into a subcutaneous pocket in the backs of mice. In vivo bioluminescence imaging, quantification of ex vivo CFU from the capsules and implants, variable-pressure scanning electron microscopy (VP-SEM), and neutrophil enhanced green fluorescent protein (EGFP) fluorescence in LysEGFP mice were employed to monitor the infection. This model was used to evaluate the efficacies of low- and high-dose cefazolin (50 and 200 mg/kg of body weight) and vancomycin (10 and 110 mg/kg) intravenous prophylaxis with or without rifampin (25 mg/kg). High-dose cefazolin and high-dose vancomycin treatment resulted in almost complete bacterial clearance, whereas both low-dose cefazolin and low-dose vancomycin reduced the in vivo and ex vivo bacterial burden only moderately. The addition of rifampin to low-dose cefazolin and vancomycin was highly effective in further reducing the CFU harvested from the implants. However, vancomycin-rifampin was more effective than cefazolin-rifampin in further reducing the CFU harvested from the surrounding tissue capsules. Future studies in humans will be required to determine whether the addition of rifampin has improved efficacy in preventing device-related infections in clinical practice.

Treatment of prosthetic joint infections often involves a two-stage exchange, with implant removal and antibiotic spacer placement followed by systemic antibiotic therapy and delayed reimplantation. However, if antibiotic therapy can be improved, one-stage exchange or implant retention may be more feasible, thereby decreasing morbidity and preserving function. In this study, a mouse model of prosthetic joint infection was used in which Staphylococcus aureus was inoculated into a knee joint containing a surgically placed metallic implant extending from the femur. This model was used to evaluate whether combination therapy of vancomycin plus rifampin has increased efficacy compared with vancomycin alone against these infections. On postoperative day 7, vancomycin with or without rifampin was administered for 6 weeks with implant retention. In vivo bioluminescence imaging, ex vivo CFU enumeration, X-ray imaging, and histologic analysis were carried out. We found that there was a marked therapeutic benefit when vancomycin was combined with rifampin compared with vancomycin alone. Taken together, our results suggest that the mouse model used could serve as a valuable in vivo preclinical model system to evaluate and compare efficacies of antibiotics and combinatory therapy for prosthetic joint infections before more extensive studies are carried out in human subjects.

A previous study reported the use of luminol for the detection of myeloperoxidase (MPO) activity using optical imaging in infiltrating neutrophils under inflammatory disease conditions. The detection is based on a photon-emitting reaction between luminol and an MPO metabolite. Because of tissue absorption and scattering, however, luminol-emitted blue light can be efficiently detected from superficial inflammatory foci only. In this study we report a chemiluminescence resonance energy transfer (CRET) methodology in which luminol-generated blue light excites nanoparticles to emit light in the near-infrared spectral range, resulting in remarkable improvement of MPO detectability in vivo. CRET caused a 37-fold increase in luminescence emission over luminol alone in detecting MPO activity in lung tissues after lipopolysaccharide challenge. We demonstrated a dependence of the chemiluminescent signal on MPO activity using MPO-deficient mice. In addition, co-administration of 4-aminobenzoic acid hydrazide (4-ABAH), an irreversible inhibitor of MPO, significantly attenuated luminescent emission from inflamed lungs. Inhibition of nitric oxide synthase with a nonspecific inhibitor, L-NAME, had no effect on luminol-mediated chemiluminescence production. Pretreatment of mice with MLN120B, a selective inhibitor of IKK-2, resulted in suppression of neutrophil infiltration to the lung tissues and reduction of MPO activity. We also demonstrated that CRET can effectively detect MPO activity at deep tissue tumor foci due to tumor development-associated neutrophil infiltration. We developed a sensitive MPO detection methodology that provides a means for visualizing and quantifying oxidative stress in deep tissue. This method is amenable to rapid evaluation of anti-inflammatory agents in animal models.

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

BackgroundRecent advances in non-invasive optical, radiographic and μCT imaging provide an opportunity to monitor biological processes longitudinally in an anatomical context. One particularly relevant application for combining these modalities is to study orthopaedic implant infections. These infections are characterized by the formation of persistent bacterial biofilms on the implanted materials, causing inflammation, periprosthetic osteolysis, osteomyelitis, and bone damage, resulting in implant loosening and failure.Methodology/principal findingsAn orthopaedic implant infection model was used in which a titanium Kirshner-wire was surgically placed in femurs of LysEGFP mice, which possess EGFP-fluorescent neutrophils, and a bioluminescent S. aureus strain (Xen29; 1×10(3) CFUs) was inoculated in the knee joint before closure. In vivo bioluminescent, fluorescent, X-ray and μCT imaging were performed on various postoperative days. The bacterial bioluminescent signals of the S. aureus-infected mice peaked on day 19, before decreasing to a basal level of light, which remained measurable for the entire 48 day experiment. Neutrophil EGFP-fluorescent signals of the S. aureus-infected mice were statistically greater than uninfected mice on days 2 and 5, but afterwards the signals for both groups approached background levels of detection. To visualize the three-dimensional location of the bacterial infection and neutrophil infiltration, a diffuse optical tomography reconstruction algorithm was used to co-register the bioluminescent and fluorescent signals with μCT images. To quantify the anatomical bone changes on the μCT images, the outer bone volume of the distal femurs were measured using a semi-automated contour based segmentation process. The outer bone volume increased through day 48, indicating that bone damage continued during the implant infection.Conclusions/significanceBioluminescent and fluorescent optical imaging was combined with X-ray and μCT imaging to provide noninvasive and longitudinal measurements of the dynamic changes in bacterial burden, neutrophil recruitment and bone damage in a mouse orthopaedic implant infection model.