Efficacy of rifampicin combination therapy against MRSA prosthetic vascular graft infections in a rat model.

Staphylococcus aureus is a major cause of prosthetic vascular graft or endograft infections (VGEIs) and the optimal choice of antibiotics is unclear. We investigated various antibiotic choices as either monotherapy or combination therapy with rifampicin against MRSA in vitro and in vivo. Fosfomycin, daptomycin and vancomycin alone or in combination with rifampicin was used against MRSA USA300 FPR3757. Each antibiotic was tested for synergism or antagonism with rifampicin in vitro, and all antibiotic regimens were tested against actively growing bacteria in media and non-growing bacteria in buffer, both as planktonic cells and in biofilms. A rat model of VGEI was used to quantify the therapeutic efficacy of antibiotics in vivo by measuring bacterial load on grafts and in spleen, liver and kidneys. In vitro, rifampicin combinations did not reveal any synergism or antagonism in relation to growth inhibition. However, quantification of bactericidal activity revealed a strong antagonistic effect, both on biofilms and planktonic cells. This effect was only observed when treating active bacteria, as all antibiotics had little or no effect on inactive cells. Only daptomycin showed some biocidal activity against inactive cells. In vivo evaluation of therapy against VGEI contrasted the in vitro results. Rifampicin significantly increased the efficacy of both daptomycin and vancomycin. The combination of daptomycin and rifampicin was by far the most effective, curing 8 of 13 infected animals. Our study demonstrates that daptomycin in combination with rifampicin shows promising potential against VGEI caused by MRSA. Furthermore, we show how in vitro evaluation of antibiotic combinations in laboratory media does not predict their therapeutic effect against VGEI in vivo, presumably due to a difference in the metabolic state of the bacteria.

Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

Rifampicin does not reduce moxifloxacin concentrations at the site of infection and may not improve treatment outcome of a one-stage exchange surgery protocol of implant-associated osteomyelitis lesions in a porcine model.

We aimed to evaluate moxifloxacin steady-state concentrations in infected bone and soft tissue and to explore the additive microbiological and pathological treatment effect of rifampicin to standard moxifloxacin treatment of implant-associated osteomyelitis (IAO). 16 pigs were included. On Day 0, IAO was induced in the proximal tibia using a susceptible Staphylococcus aureus strain. On Day 7, the pigs underwent one-stage exchange surgery of the IAO lesions and were randomized to receive seven days of intravenous antibiotic treatment of either rifampicin combined with moxifloxacin or moxifloxacin monotherapy. On Day 14, microdialysis was applied for continuous sampling (8 h) of moxifloxacin concentrations. Microbiological, macroscopical pathology, and histopathological analyses were performed postmortem. Steady-state moxifloxacin area under the concentration-time curve was lower in the combination therapy group in plasma (total) and subcutaneous tissue compartments (infected and noninfected) (p < 0.04), while no differences were found in bone compartments. No additional treatment effect of rifampicin to moxifloxacin treatment was found (p = 0.57). Conclusive, additive rifampicin treatment does not reduce moxifloxacin concentrations at the infection site. Rifampicin treatment may not be necessary in a one-stage exchange treatment of IAO. However, our sample size and treatment period may have been too small and short to reveal true clinical differences.

Fibrinolytic and antibiotic treatment of prosthetic vascular graft infections in a novel rat model.

OBJECTIVES: We developed a rat model of prosthetic vascular graft infection to assess, whether the fibrinolytic tissue plasminogen activator (tPA) could increase the efficacy of antibiotic therapy. MATERIALS AND METHODS: Rats were implanted a polyethylene graft in the common carotid artery, pre-inoculated with approx. 6 log10 colony forming units (CFU) of methicillin resistant Staphylococcus aureus. Ten days after surgery, rats were randomized to either: 0.9% NaCl (n = 8), vancomycin (n = 8), vancomycin + tPA (n = 8), vancomycin + rifampicin (n = 18) or vancomycin + rifampicin + tPA (n = 18). Treatment duration was seven days. Approximately 36 hours after the end of treatment, the rats were euthanized, and grafts and organs were harvested for CFU enumeration. RESULTS: All animals in the control group had significantly higher CFU at the time of euthanization compared to bacterial load found on the grafts prior to inoculation (6.45 vs. 4.36 mean log10 CFU/mL, p = 0.0011), and both the procedure and infection were well tolerated. Vancomycin and rifampicin treatment were superior to monotherapy with vancomycin, as it lead to a marked decrease in median bacterial load on the grafts (3.50 vs. 6.56 log10 CFU/mL, p = 0.0016). The addition of tPA to vancomycin and rifampicin combination treatment did not show a further decrease in bacterial load (4.078 vs. 3.50 log10 CFU/mL, p = 0.26). The cure rate was 16% in the vancomycin + rifampicin group vs. 37.5% cure rate in the vancomycin + rifampicin + tPA group. Whilst interesting, this trend was not significant at our sample size (p = 0.24). CONCLUSION: We developed the first functional model of an arterial prosthetic vascular graft infection in rats. Antibiotic combination therapy with vancomycin and rifampicin was superior to vancomycin monotherapy, and the addition of tPA did not significantly reduce bacterial load, nor significantly increase cure rate.

Shotgun sequencing of sonication fluid for the diagnosis of orthopaedic implant-associated infections with Cutibacterium acnes as suspected causative agent.

Orthopaedic implant-associated infections (OIAIs) due to Cutibacterium acnes can be difficult to diagnose. The aim of this pilot study was to determine if metagenomic next-generation sequencing (mNGS) can provide additional information to improve the diagnosis of C. acnes OIAIs. mNGS was performed on sonication fluid (SF) specimens derived from 24 implants. These were divided into three groups, based on culture results: group I, culture-negative (n = 4); group II, culture-positive for C. acnes (n = 10); and group III, culture-positive for other bacteria (n = 10). In group I, sequence reads from C. acnes were detected in only one SF sample, originating from a suspected case of OIAIs, which was SF and tissue culture-negative. In group II, C. acnes sequences were detected in 7/10 samples. In group III, C. acnes sequence reads were found in 5/10 samples, in addition to sequence reads that matched the bacterial species identified by culture. These samples could represent polymicrobial infections that were missed by culture. Taken together, mNGS was able to detect C. acnes DNA in more samples compared to culture and could be used to identify cases of suspected C. acnes OIAIs, in particular regarding possible polymicrobial infections, where the growth of C. acnes might be compromised due to a fast-growing bacterial species. However, since SF specimens are usually low-biomass samples, mNGS is prone to DNA contamination, possibly introduced during DNA extraction or sequencing procedures. Thus, it is advisable to set a sequence read count threshold, taking into account project- and NGS-specific criteria.

Guideline for management of septic arthritis in native joints (SANJO).

This clinical guideline is intended for use by orthopedic surgeons and physicians who care for patients with possible or documented septic arthritis of a native joint (SANJO). It includes evidence and opinion-based recommendations for the diagnosis and management of patients with SANJO.

JMM Profile: Staphylococcus epidermidis.

Staphylococcus epidermidis is the most abundant commensal bacterium of human skin. Despite protecting us from foreign invaders, S. epidermidis itself exploits human vulnerability when given the opportunity. Such opportunities arise when patients are immunocompromised or when biomedical implants present an opportunity to colonize the surface and form biofilms. S. epidermidis is one of the most frequently isolated organisms from implanted devices and from bloodstream infections. However, S. epidermidis infections are often recalcitrant to antibiotics because of biofilm-associated antibiotic tolerance. Furthermore, the emergence and spread of nearly pan-resistant strains is a considerable health concern. Symptoms can be subclinical, making diagnosis challenging, and treatment with antibiotics is inefficient. For now, infection prevention remains the best strategy available.

Amplicon-Based Next-Generation Sequencing as a Diagnostic Tool for the Detection of Phylotypes of Cutibacterium acnes in Orthopedic Implant-Associated Infections.

The diagnosis of orthopedic implant-associated infections (OIAIs) caused by the slow-growing anaerobic bacterium Cutibacterium acnes is challenging. The mild clinical presentations of this low-virulent bacterium along with its ubiquitous presence on human skin and human-dominated environments often make it difficult to differentiate true infection from contamination. Previous studies have applied C. acnes phylotyping as a potential avenue to distinguish contamination from infection; several studies reported a prevalence of phylotypes IB [corresponding to type H in the single-locus sequence typing (SLST) scheme] and II (SLST type K) in OIAIs, while a few others found phylotype IA1 (more specifically SLST type A) to be abundant. However, phylotype determination has mainly been done in a culture-dependent manner on randomly selected C. acnes isolates. Here, we used a culture-independent amplicon-based next-generation sequencing (aNGS) approach to determine the presence and relative abundances of C. acnes phylotypes in clinical OIAI specimens. As amplicon, the SLST target was used, a genomic fragment that is present in all C. acnes strains known to date. The aNGS approach was applied to 30 sonication fluid (SF) samples obtained from implants removed during revision surgeries, including 17 C. acnes culture-positive and 13 culture-negative SF specimens. In 53% of the culture-positive samples, SLST types were identified: relative abundances were highest for K-type C. acnes, followed by H- and D-type C. acnes. Other types, including A- and C-type C. acnes that are more prevalent on human skin, had low relative abundances. The aNGS results were compared with, and confirmed by a culture-dependent approach, which included the isolation, whole genome sequencing (WGS) and phylotyping of 36 strains of C. acnes obtained from these SF samples. Besides serving as a powerful adjunct to identify C. acnes phylotypes, the aNGS approach could also distinguish mono- from heterotypic infections, i.e., infections caused by more than one phylotype of C. acnes: in eight out of nine culture-positive SF samples multiple C. acnes types were detected. We propose that the aNGS approach, along with the patient's clinical information, tissue and SF cultures and WGS, could help differentiate C. acnes contamination from true infection.

Tibial bone and soft-tissue concentrations following combination therapy with vancomycin and meropenem - evaluated by microdialysis in a porcine model : should patients with open fractures have higher doses of antibiotics?

AIMS: Prompt and sufficient broad-spectrum empirical antibiotic treatment is key to preventing infection following open tibial fractures. Succeeding co-administration, we dynamically assessed the time for which vancomycin and meropenem concentrations were above relevant epidemiological cut-off (ECOFF) minimal inhibitory concentrations (T > MIC) in tibial compartments for the bacteria most frequently encountered in open fractures. Low and high MIC targets were applied: 1 and 4 µg/ml for vancomycin, and 0.125 and 2 µg/ml for meropenem. METHODS: Eight pigs received a single dose of 1,000 mg vancomycin and 1,000 mg meropenem simultaneously over 100 minutes and 10 minutes, respectively. Microdialysis catheters were placed for sampling over eight hours in tibial cancellous bone, cortical bone, and adjacent subcutaneous adipose tissue. Venous blood samples were collected as references. RESULTS: Across the targeted ECOFF values, vancomycin displayed longer T > MIC in all the investigated compartments in comparison to meropenem. For both drugs, cortical bone exhibited the shortest T > MIC. For the low MIC targets and across compartments, mean T > MIC ranged between 208 and 449 minutes (46% to 100%) for vancomycin and between 189 and 406 minutes (42% to 90%) for meropenem. For the high MIC targets, mean T > MIC ranged between 30 and 446 minutes (7% to 99%) for vancomycin and between 45 and 181 minutes (10% to 40%) for meropenem. CONCLUSION: The differences in the T > MIC between the low and high targets illustrate how the interpretation of these results is highly susceptible to the defined MIC target. To encompass any trauma, contamination, or individual tissue differences, a more aggressive dosing approach may be considered to achieve longer T > MIC in all the exposed tissues, and thereby lower the risk of acquiring an infection after open tibial fractures. Cite this article: Bone Joint Res 2022;11(2):112-120.

Moxifloxacin Concentrations in the Knee Joint, Tibial Bone, and Soft Tissue When Combined with Rifampicin: A Randomized Porcine Microdialysis Study.

BACKGROUND: Peri and postoperative antibiotics are key adjuvant treatment tools in the management of periprosthetic joint infection (PJI). The aim of this study was to evaluate the effect of rifampicin on the area under the moxifloxacin concentration-time curve from 0 to 24 hours (AUC0-24) in the synovial fluid of the knee joint, tibial bone, and adjacent subcutaneous tissue under steady-state conditions using microdialysis in a porcine model. METHODS: Twenty female pigs were randomized to receive oral treatment with moxifloxacin monotherapy (Group A, n = 10) of 400 mg once daily for 3 days or a combination therapy (Group B, n = 10) of 400 mg of moxifloxacin once daily for 3 days and 450 mg of rifampicin twice daily for 7 days. Microdialysis was used for sampling the synovial fluid of the knee joint, tibial cancellous and cortical bone, and adjacent subcutaneous tissues. Plasma samples were taken as a reference. Measurements were obtained for 24 hours. RESULTS: Coadministration of moxifloxacin and rifampicin resulted in reductions of the moxifloxacin AUC0-24 in all targeted tissue compartments by 67% to 85% (p < 0.05). The corresponding change in plasma was 20% (p = 0.49). For both groups, the tissue penetration (the ratio of tissue free fraction AUC0-24 to plasma free fraction AUC0-24 [fAUCtissue/fAUCplasma]) was incomplete in all investigated compartments. The highest moxifloxacin tissue penetration was in the knee joint synovial fluid: 0.59 (Group A) and 0.24 (Group B). The lowest tissue penetration was in the cortical bone: 0.17 (Group A) and 0.03 (Group B). CONCLUSIONS: We found a significant reduction of the moxifloxacin concentration, expressed as the AUC0-24, in tissues relevant to acute PJI treatment when coadministered with rifampicin. CLINICAL RELEVANCE: The concentrations within the targeted tissue compartments were reduced significantly more than the concentrations in plasma, which may be particularly important as plasma concentrations are used in clinical practice to assess moxifloxacin treatment sufficiency.

Diagnosis of orthopaedic-implant-associated infections caused by slow-growing Gram-positive anaerobic bacteria - a clinical perspective.

Slow-growing Gram-positive anaerobic bacteria (SGAB) such as Cutibacterium acnes are increasingly recognized as causative agents of implant-associated infections (IAIs) in orthopaedic surgeries. SGAB IAIs are difficult to diagnose because of their non-specific clinical and laboratory findings as well as the fastidious growth conditions required by these bacteria. A high degree of clinical suspicion and awareness of the various available diagnostic methods is therefore important. This review gives an overview of the current knowledge regarding SGAB IAI, providing details about clinical features and available diagnostic methodologies. In recent years, new methods for the diagnosis of IAI were developed, but there is limited knowledge about their usefulness in SGAB IAI. Further studies are required to determine the ideal diagnostic methodology to identify these infections so that they are not overlooked and mistakenly classified as aseptic failure.

Genome Sequence of Staphylococcus epidermidis AUH4567, a Clinical Isolate from an Infected Central Venous Catheter.

Staphylococcus epidermidis is a common cause of implant-associated infections, and this is related to its ability to form biofilms. Strain-to-strain variability in biofilm formation is likely caused by genetic differences. Here, we present a draft genome of S. epidermidis AUH4567, which was isolated from a central venous catheter infection.

Host factors abolish the need for polysaccharides and extracellular matrix-binding protein in Staphylococcus epidermidis biofilm formation.

Introduction. Staphylococcus epidermidis is predominant in implant-associated infections due to its capability to form biofilms. It can deploy several strategies for biofilm development using either polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA) and/or proteins, such as the extracellular matrix-binding protein (Embp).Hypothesis/Gap Statement. We hypothesize that the dichotomic regulation of S. epidermidis adhesins is linked to whether it is inside a host or not, and that in vitro biofilm investigations in laboratory media may not reflect actual biofilms in vivo.Aim. We address the importance of PIA and Embp in biofilm grown in 'humanized' media to understand if these components play different roles in biofilm formation under conditions where bacteria can incorporate host proteins in the biofilm matrix.Methodology. S. epidermidis 1585 WT (deficient in icaADBC), and derivative strains that either lack embp, express embp from an inducible promotor, or express icaADBC from a plasmid, were cultivated in standard laboratory media, or in media with human plasma or serum. The amount, structure, elasticity and antimicrobial penetration of biofilms was quantified to describe structural differences caused by the different matrix components and growth conditions. Finally, we quantified the initiation of biofilms as suspended aggregates in response to host factors to determine how quickly the cells aggregate in response to the host environment and reach a size that protects them from phagocytosis.Results. S. epidermidis 1585 required polysaccharides to form biofilm in laboratory media. However, these observations were not representative of the biofilm phenotype in the presence of human plasma. If human plasma were present, polysaccharides and Embp were redundant for biofilm formation. Biofilms formed in human plasma were loosely attached and existed mostly as suspended aggregates. Aggregation occurred after 2 h of exposing cells to plasma or serum. Despite stark differences in the amount and composition of biofilms formed by polysaccharide-producing and Embp-producing strains in different media, there were no differences in vancomycin penetration or susceptibility.Conclusion. We suggest that the assumed importance of polysaccharides for biofilm formation is an artefact from studying biofilms in laboratory media void of human matrix components. The cell-cell aggregation of S. epidermidis can be activated by host factors without relying on either of the major adhesins, PIA and Embp, indicating a need to revisit the basic question of how S. epidermidis deploys self-produced and host-derived matrix components to form antibiotic-tolerant biofilms in vivo.

[Management of Staphylococcus aureus bacteraemia].

In this review, we discuss Staphylococcus aureus bac-teraemia (SAB), which is a frequent and severe condition associated with high morbidity and mortality. Despite these facts, high-quality data on diagnostic approach, treatment and management of SAB remain scarce. Consequently, evidence-based guidelines concerning antibiotic therapy including the optimal choice of antibiotic drug, route of administration and treatment duration are not available. Thus, controlled clinical trials are urgently needed to increase evidence and to potentially improve the outcome for patients with SAB.

Ultra-dense polymer brush coating reduces Staphylococcus epidermidis biofilms on medical implants and improves antibiotic treatment outcome.

Staphylococcal biofilm formation is a severe complication of medical implants, leading to high antibiotic tolerance and treatment failure. Ultra-dense poly(ethylene glycol) (udPEG) coating resists adsorption of proteins, polysaccharides and extracellular DNA. It is therefore uniquely resistant to attachment by Staphylococcus epidermidis, which remains loosely adhered to the surface. Our aim was to determine if S. epidermidis remains susceptible to antibiotics when adhering to udPEG, and if udPEG coatings can improve the treatment outcome for implant-associated infections. We tested the in vitro efficacy of vancomycin treatment on recently adhered S. epidermidis AUH4567 on udPEG, conventional PEG or titanium surfaces using live/dead staining and microscopy. udPEG was then applied to titanium implants and inserted subcutaneously in mice and inoculated with S. epidermidis to induce infection. Mice were given antibiotic prophylaxis or a short antibiotic treatment. One group was given immunosuppressive therapy. After five days, implants and surrounding tissue were harvested for CFU enumeration. Only few S. epidermidis cells adhered to udPEG compared to conventional PEG and uncoated titanium, and a much lower fraction of cells on udPEG survived antibiotic treatment in vitro. In vivo, the bacterial load on implants in mice receiving vancomycin treatment was significantly lower on udPEG-coated compared to uncoated implants, also in neutropenic mice. Our results suggest that the improved outcome results from the coating's anti-adhesive properties that leads to less biofilm and increased efficacy of antibiotic treatment. Thus, the combination of udPEG with antibiotics is a promising strategy to prevent acute implant-associated infections that arise due to perioperative contaminations. STATEMENT OF SIGNIFICANCE: Infections of medical implants is an ever-present danger. Here, bacteria develop biofilms that cannot be eradicated with antibiotics. By using an ultra-dense polymer-brush coating (udPEG), bacterial attachment and the subsequent biofilm formation can be reduced, resulting in increased antibiotic susceptibility of bacteria surrounding the implant. udPEG combined with antibiotics proved to significantly reduce bacteria on implants inserted into mice, in our animal model. As the coating is not antibacterial per se, it does not induce antimicrobial resistance and its effect is independent of the bacterial species. Our results are encouraging for the prospect of preventing and treating implant-associated infections that arise due to perioperative contaminations.

Hyperbaric Oxygen Therapy is Ineffective as an Adjuvant to Daptomycin with Rifampicin Treatment in a Murine Model of Staphylococcus aureus in Implant-Associated Osteomyelitis.

Implant-associated infections caused by bacterial biofilms are difficult to treat. Surgical intervention is often necessary to cure the patient, as the antibiotic recalcitrance of biofilms renders them untreatable with conventional antibiotics. Intermittent hyperbaric oxygen treatment (HBOT) has been proposed as an adjuvant to conventional antibiotic treatment and it has been speculated that combining HBOT with antibiotics could improve treatment outcomes for biofilm infections. In this study we addressed whether HBOT could improve treatment outcomes of daptomycin and rifampicin combination therapy. The effect of HBOT on the treatment outcomes of daptomycin and rifampicin against implant-associated osteomyelitis was quantified in a murine model. In total, 80 mice were randomized into two groups receiving antibiotics, either alone or in combination with daily intermittent HBOT (304 kPa for 60 min) following injection of antibiotics. Treatment was initiated 11 days after animals were infected with Staphylococcus aureus and treatment duration was 14 days. We found that HBOT did not improve the cure rate and did not reduce the bacterial load on the implant surface or in the surrounding tissue. Cure rates of daptomycin + rifampicin were 40% in infected tibias and 75% for implants while cure rates for HBOT-daptomycin + rifampicin were 50% and 85%, respectively, which were not significantly higher (Fisher's exact test). While it is encouraging that the combination of daptomycin and rifampicin is very effective, our study demonstrates that this efficacy cannot be improved by adjuvant HBOT.

Cholinergic PET imaging in infections and inflammation using 11C-donepezil and 18F-FEOBV.

INTRODUCTION: Immune cells utilize acetylcholine as a paracrine-signaling molecule. Many white blood cells express components of the cholinergic signaling pathway, and these are up-regulated when immune cells are activated. However, in vivo molecular imaging of cholinergic signaling in the context of inflammation has not previously been investigated. METHODS: We performed positron emission tomography (PET) using the glucose analogue 18F-FDG, and 11C-donepezil and 18F-FEOBV, markers of acetylcholinesterase and the vesicular acetylcholine transporter, respectively. Mice were inoculated subcutaneously with Staphylococcus aureus, and PET scanned at 24, 72, 120, and 144 h post-inoculation. Four pigs with post-operative abscesses were also imaged. Finally, we present initial data from human patients with infections, inflammation, and renal and lung cancer. RESULTS: In mice, the FDG uptake in abscesses peaked at 24 h and remained stable. The 11C-donepezil and 18F-FEOBV uptake displayed progressive increase, and at 120-144 h was nearly at the FDG level. Moderate 11C-donepezil and slightly lower 18F-FEOBV uptake were seen in pig abscesses. PCR analyses suggested that the 11C-donepezil signal in inflammatory cells is derived from both acetylcholinesterase and sigma-1 receptors. In humans, very high 11C-donepezil uptake was seen in a lobar pneumonia and in peri-tumoral inflammation surrounding a non-small cell lung carcinoma, markedly superseding the 18F-FDG uptake in the inflammation. In a renal clear cell carcinoma no 11C-donepezil uptake was seen. DISCUSSION: The time course of cholinergic tracer accumulation in murine abscesses was considerably different from 18F-FDG, demonstrating in the 11C-donepezil and 18F-FEOBV image distinct aspects of immune modulation. Preliminary data in humans strongly suggest that 11C-donepezil can exhibit more intense accumulation than 18F-FDG at sites of chronic inflammation. Cholinergic PET imaging may therefore have potential applications for basic research into cholinergic mechanisms of immune modulation, but also clinical applications for diagnosing infections, inflammatory disorders, and cancer inflammation.

Streptokinase Treatment Reverses Biofilm-Associated Antibiotic Resistance in Staphylococcus aureus.

Biofilms formed by Staphylococcus aureus is a serious complication to the use of medical implants. A central part of the pathogenesis relies on S. aureus' ability to adhere to host extracellular matrix proteins, which adsorb to medical implants and stimulate biofilm formation. Being coagulase positive, S. aureus furthermore induces formation of fibrin fibers from fibrinogen in the blood. Consequently, we hypothesized that fibrin is a key component of the extracellular matrix of S. aureus biofilms under in vivo conditions, and that the recalcitrance of biofilm infections can be overcome by combining antibiotic treatment with a fibrinolytic drug. We quantified S. aureus USA300 biofilms grown on peg-lids in brain heart infusion (BHI) broth with 0%-50% human plasma. Young (2 h) and mature (24 h) biofilms were then treated with streptokinase to determine if this lead to dispersal. Then, the minimal biofilm eradication concentration (MBEC) of 24 h old biofilms was measured for vancomycin and daptomycin alone or in combination with 10 µg/mL rifampicin in the presence or absence of streptokinase in the antibiotic treatment step. Finally, biofilms were visualized by confocal laser scanning microscopy. Addition of human plasma stimulated biofilm formation in BHI in a dose-dependent manner, and biofilms could be partially dispersed by streptokinase. The biofilms could be eradicated with physiologically relevant concentrations of streptokinase in combination with rifampicin and vancomycin or daptomycin, which are commonly used antibiotics for treatment of S. aureus infections. Fibronolytic drugs have been used to treat thromboembolic events for decades, and our findings suggest that their use against biofilm infections has the potential to improve the efficacy of antibiotics in treatment of S. aureus biofilm infections.

Rifampicin-containing combinations are superior to combinations of vancomycin, linezolid and daptomycin against Staphylococcus aureus biofilm infection in vivo and in vitro.

Susceptibility to antibiotics is dramatically reduced when bacteria form biofilms. In clinical settings this has a profound impact on treatment of implant-associated infections, as these are characterized by biofilm formation. Current routine susceptibility testing of microorganisms from infected implants does not reflect the actual susceptibility, and the optimal antibiotic strategy for treating implant-associated infections is not established. In this study of biofilm formation in implant-associated osteomyelitis, we compared thein vitroandin vivoefficacy of selected antibiotics alone and in combination againstStaphylococcus aureus.We tested vancomycin, linezolid, daptomycin and tigecycline alone and in combination with rifampicin, vancomycin, linezolid and daptomycin againstS. aureusIn vitro, biofilm formation dramatically reduced susceptibility by a factor of 500-2000.In vivo, antibiotic combinations were tested in a murine model of implant-associated osteomyelitis. Mice were infected by inserting implants colonized withS. aureustrough their tibia. After 11 days, the animals were divided into different groups (five animals/group) and given 14 days of antibiotic therapy. All antibiotics resulted in a reduced bacterial load in the infected bone surrounding the implant. Overall, the most effective antibiotic combinations contained rifampicin. Combinations containing two non-rifampicin antibiotics were not more active than single drugs.