Implant-associated Osteomyelitis Research Articles

Bacterial micro-aggregates as inoculum in animal models of implant-associated infections

Is it time to rethink the inoculum of animal models of implant-associated infections (IAI)? Traditionally, animal models of IAI are based on inoculation with metabolically active overnight cultures of planktonic bacteria or pre-grown surface-attached biofilms. However, such inoculums do not mimic the clinical initiation of IAI. Therefore, the present study aimed to develop a clinically relevant inoculum of low metabolic micro-aggregated bacteria. The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth (TSB) for seven days to facilitate the formation of low metabolic micro-aggregates. Subsequently, the aggregated culture underwent filtration using cell strainers of different pore sizes to separate micro-aggregates. Light microscopy was used to evaluate the aggregate formation and size in the different fractions, while isothermal microcalorimetry was used to disclose a low metabolic activity. The micro-aggregate fraction obtained with filter size 5–15 μm (actual measured mean size 32 μm) was used as inoculum in a porcine implant-associated osteomyelitis (IAO) model and compared to a standard overnight planktonic inoculum and a sham inoculum of 0.9 % saline. The micro-aggregate and planktonic inoculums caused IAO with the re-isolation of S. aureus from soft tissues, bones, and implants. However, compared to their planktonic counterpart, neither of the micro-aggregate inoculated animals showed signs of osteomyelitis, i.e., sequester, osteolysis, and pus at gross inspection. Furthermore, inoculation with low metabolic micro-aggregates resulted in a strong healing response with pronounced osteoid formation, comparable to sham animals. In conclusion, the formation and separation of low metabolic bacterial micro-aggregates into various size fractions is possible, however, planktonic bacteria were still seen in all size fractions. Inoculation with micro-aggregates caused a less-aggressive osteomyelitis i.e. combination of infected tissue and strong healing response. Therefore, the use of low metabolic micro-aggregates could be a relevant inoculum for animal models of less-aggressive and thereby slower developing IAI and add in to our understanding of the host-implant-bacteria interactions in slow-onset low-grade infections.

A Self-Transformed N-Chlorinated ε-Polylysine Coating Endows Titanium Implants with Programmed Integration of Robust Antibacterial and Pro-Osteogenic Abilities

Implant-associated infection and poor osseointegration are the main causes of orthopedic implantation failure. For a long-term successful implantation, simultaneously introducing prevention of infection and promotion of osteogenesis into implants is highly desired but remains a grand challenge. Inspired by the human immune mechanism of tissue repair under bacterial infection, we propose a self-transformed strategy for the modification of titanium with porous N-chlorinated ε-polylysine coating (Ti-PL-NCl) through pore-making, surface grafting and chlorination. The as-prepared Ti-PL-NCl exhibits an excellent antibacterial activity against S. aureus and E. coli in the presence of N-chloramine in the early stage, with an antibacterial rate as high as 95.8% and 99.4%, respectively. With the consumption of oxidative chlorine, the N─Cl groups of Ti-PL-NCl are transformed into N─H groups with moderate antibacterial yet strong pro-osteogenic abilities. The self-transformed N-chlorinated ε-polylysine coating enables Ti-PL-NCl to achieve long-lasting antibacterial activity as well as enhanced pro-osteogenesis, thus providing adaptive bioactivities for bone-implant integration. Rat implant-associated osteomyelitis model confirms that Ti-PL-NCl has excellent efficacy of infection prevention and osseointegration promotion. This work may provide a new paradigm of designing multifunctional implants to achieve long-term successful implantation.

Eradication of Staphylococcus aureus in Implant-Associated Osteomyelitis by an Injectable in situ-Forming Depot Antibiotics Delivery System.

Bone infections from Staphylococcus aureus are notoriously difficult to treat and have high recurrence rates. Local antibiotic delivery systems hold the potential to achieve high in situ antibiotic concentrations, which are otherwise challenging to achieve via systemic administration. Existing solutions have been shown to confer suboptimal drug release and distribution. Here we present and evaluate an injectable in situ-forming depot system termed CarboCell. The CarboCell technology provides sustained and tuneable release of local high-dose antibiotics. CarboCell formulations of levofloxacin or clindamycin with or without antimicrobial adjuvants cis-2-decenoic acid or cis-11-methyl-2-dodecenoic acid were tested in experimental rodent and porcine implant-associated osteomyelitis models. In the porcine models, debridement and treatment with CarboCell-formulated antibiotics was carried out without systemic antibiotic administration. The bacterial burden was determined by quantitative bacteriology. CarboCell formulations eliminated S. aureus in infected implant rat models. In the translational implant-associated pig model, surgical debridement, and injection of clindamycin-releasing CarboCell formulations resulted in pathogen-free bone tissues and implants in 9/12, and full eradication in 5/12 pigs. Sustained release of antimicrobial agents mediated by the CarboCell technology demonstrated promising therapeutic efficacy in challenging translational models and may be beneficial in combination with the current standard of care.

Remodeling Microenvironment for Implant-Associated Osteomyelitis by Dual Metal Peroxide.

Implant-associated osteomyelitis (IAOM) is characterized by bone infection and destruction; current therapy of antibiotic treatment and surgical debridement often results in drug resistance and bone defect. It is challenging to develop an antibiotic-free bactericidal and osteogenic-enhanced strategy for IAOM. Herein, an IAOM-tailored antibacterial and osteoinductive composite of copper (Cu)-strontium (Sr) peroxide nanoparticles (CSp NPs), encapsulated in polyethylene glycol diacrylate (PEGDA) (CSp@PEGDA), is designed. The dual functional CSp NPs display hydrogen peroxide (H2 O2 ) self-supplying and Fenton catalytic Cu2+ ions' release, generating plenty of hydroxyl radical (• OH) in a pH-responsive manner for bacterial killing, while the released Sr2+ promotes the in vitro osteogenicity regarding cell proliferation, alkaline phosphatase activity, extracellular matrix calcification, and osteo-associated genes expression. The integration of Cu2+ and Sr2+ in CSp NPs together with the coated PEGDA hydrogel ensures the stable and sustainable ion release during short- and long-term periods. Benefitted from the injectablity and photo-crosslink ability, CSp@PEGDA is able to thoroughly fill the infectious site and gelate in situ for bacterial elimination and bone regeneration, which is verified through in vivo evaluation using a clinical-simulating IAOM mouse model. These favorable abilities of CSp@PEGDA precisely meet the multiple therapeutic needs and pave a promising way for implant-associated osteomyelitis treatment.

Preventing septic implant failures in osteoporotic hip fractures using antibiotic-loaded functionalized nanocement

Osteoporosis is a prevalent skeletal disorder and the most common cause of debilitating hip fractures, primarily affecting post-menopausal women and elderly people. Implants used to stabilize osteoporotic fragility fractures are susceptible to bacterial infections, which further deteriorate the implantationsite. In the current study, we developed a nanocement-based treatment for tackling hip implant infection in an osteoporotic rat model. The development of osteoporosis was confirmed by an 80% decrease in serum estradiol levels and a further reduced T-score value of −4.170 ± 0.411 for femur neck canal. Further, an infected K-wire was implanted in femoral neck canal to induce implant-associated osteomyelitis. In order to combat infection and promote bone healing, it was followed by debridement and implantation of rifampicin-loaded nanocement functionalized with various combinations of bone marrow MSCs (bMSC) exosomes, bone morphogenetic protein-2 (BMP-2) and zoledronic acid (ZA). The findings of this study demonstrated the potential of exosomes as an alternative for reducingBMP-2 dosage, and the treatment promoted successful bone regeneration withenhanced mechanical strength, and a significant decrease in bacterial load. Improved bone remodeling and matrix deposition at the implantation site were confirmed by DXA, micro-CT, and histology. Further, immunohistochemical staining showed increased expression of collagen I and CD31, which indicated bone matrix deposition and vascularization, respectively, while reduced expression of α-SMA and NOS2 confirmed a reduction in fibrosis and inflammation, respectively. Overall, our one-step treatment strategy has the potential to prevent implant-associated bone infection in osteoporotic hip fractures, promote bone formation, and enhance the mechanical strength of osteoporotic bone, thereby preventing subsequent secondary fractures.

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.

Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice.

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Δpvl), or complemented mutant (Δpvl+pvl). Three days post-surgery, Δpvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Δpvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR+ monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Δpvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

Suppression of Staphylococcus aureus biofilm formation under a short-term impact of low-intensity direct current in vitro and in a rat model of implant-associated osteomyelitis.

We investigated the effect of short-term low-intensity direct current (LIDC) on Staphylococcus aureus. The reference strain of S. aureus was used. Experiments were performed in agar culture and on a model of rat's femur osteomyelitis. K-wires were used as electrodes. The exposure to LIDC of 150 μA continued for one minute. In vitro exposure was performed once. In vivo group 1 was a control group. Osteomyelitis was modeled in three groups but only groups 3 and 4 were exposed to LIDC four times: either from day 1 or from day 7 post-surgery. The effect was evaluated on day 21. Microbiological, histological, scanning electron, and light microscopy methods were used for evaluation of the LIDC effect. Bacteria diameter, oblongness, and division increased 15 min after LIDC exposure in the culture around the cathode. After 24 hr, the amount of exomatrix was lower than in the control test, and the cell diameter and roundness increased. Similar changes around the anode were less pronounced. In vivo, biofilm formation on the intramedullary wire cathode was suppressed in group 3. In group 4, detachment and destruction of the biofilm were observed. The formation of S. aureus microcolonies was suppressed, and the adhesion of fibroblasts and immune cells was activated. LIDC did not stop the development of the osteomyelitis process. Short-term exposure to LIDC suppresses S. aureus biofilm formation on the implant cathode surface in the acute and early postoperative period but does not have an impact on the development of osteomyelitis.

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.

Evidence of bisphosphonate-conjugated sitafloxacin eradication of established methicillin-resistant S. aureus infection with osseointegration in murine models of implant-associated osteomyelitis

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve “target-and-release” drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (hydroxybisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

Antibody-Drug Conjugates to Treat Bacterial Biofilms via Targeting and Extracellular Drug Release.

The treatment of implant-associated bacterial infections and biofilms is an urgent medical need and a grand challenge because biofilms protect bacteria from the immune system and harbor antibiotic-tolerant persister cells. This need is addressed herein through an engineering of antibody-drug conjugates (ADCs) that contain an anti-neoplastic drug mitomycin C, which is also a potent antimicrobial against biofilms. The ADCs designed herein release the conjugated drug without cell entry, via a novel mechanism of drug release which likely involves an interaction of ADC with the thiols on the bacterial cell surface. ADCs targeted toward bacteria are superior by the afforded antimicrobial effects compared to the non-specific counterpart, in suspension and within biofilms, in vitro, and in an implant-associated murine osteomyelitis model in vivo. The results are important in developing ADC for a new area of application with a significant translational potential, and in addressing an urgent medical need of designing a treatment of bacterial biofilms.

Reduced angiogenesis and delayed endochondral ossification in CD163-/- mice highlights a role of M2 macrophages during bone fracture repair.

While recent studies showed that macrophages are critical for bone fracture healing, and lack of M2 macrophages have been implicated in models of delayed union, functional roles for specific M2 receptors have yet to be defined. Moreover, the M2 scavenger receptor CD163 has been identified as a target to inhibit sepsis following implant-associated osteomyelitis, but potential adverse effects on bone healing during blockage therapy have yet to be explored. Thus, we investigated fracture healing in C57BL/6 versus CD163-/- mice using a well-established closed, stabilized, mid-diaphyseal femur fracture model. While gross fracture healing in CD163-/- mice was similar to that of C57BL/6, plain radiographs revealed persistent fracture gaps in the mutant mice on Day 14, which resolved by Day 21. Consistently, 3D vascular micro-CT demonstrated delayed union on Day 21, with reduced bone volume (74%, 61%, and 49%) and vasculature (40%, 40%, and 18%) compared to C57BL/6 on Days 10, 14, and 21 postfracture, respectively (p < 0.01). Histology confirmed large amounts of persistent cartilage in CD163-/- versus C57BL/6 fracture callus on Days 7 and 10 that resolves over time, and immunohistochemistry demonstrated deficiencies in CD206+ M2 macrophages. Torsion testing of the fractures confirmed the delayed early union in CD163-/- femurs, which display decreased yield torque on Day 21, and a decreased rigidity with a commensurate increase in rotation at yield on Day 28 (p < 0.01). Collectively, these results demonstrate that CD163 is required for normal angiogenesis, callus formation, and bone remodeling during fracture healing, and raise potential concerns about CD163 blockade therapy.

Steady-State Piperacillin Concentrations in the Proximity of an Orthopedic Implant: A Microdialysis Porcine Study

Implant-associated osteomyelitis is one of the most feared complications following orthopedic surgery. Although the risk is low, sufficient antibiotic protection of the implant surface is important. The aim of this study was to assess steady-state piperacillin concentrations in the proximity of an orthopedic implant. Time above the minimal inhibitory concentration (fT>MIC) was evaluated for MIC of 8 (low target) and 16 μg/mL (high target). Six female pigs received an intravenous bolus infusion of 4 g/0.5 g piperacillin/tazobactam over 30 min every 6 h. Steady state was assumed achieved in the third dosing interval (12-18 h). Microdialysis catheters were placed in a cannulated screw in the proximal tibial cancellous bone, in cancellous bone next to the screw, and in cancellous bone on the contralateral tibia. Dialysates were collected from time 12 to 18 h and plasma samples were collected as reference. For the low piperacillin target (8 µg/mL), comparable mean fT>MIC across all the investigated compartments (mean range: 54-74%) was found. For the high target (16 µg/mL), fT>MIC was shorter inside the cannulated screw (mean: 16%) than in the cancellous bone next to the screw and plasma (mean range: 49-54%), and similar between the two cancellous bone compartments. To reach more aggressive piperacillin fT>MIC targets in relation to the implant, alternative dosing regimens such as continuous infusion may be considered.

The efficacy of vancomycin-loaded biphasic calcium phosphate bone substitute in the promotion of new bone growth and the prevention of postoperative infection.

Background: Due to the increasing need for suitable alternatives to bone grafts, artificial bones made of biphasic calcium phosphate (BCP) are currently being extensively researched. These porous bone substitutes have also demonstrated considerable incorporation with the host bone, and new bone is able to grow within the porous structure. They therefore offer a potential therapeutic approach for bone defects. Methods: Vancomycin-loaded Bicera™, a BCP bone substitute, was investigated in order to prevent implant-associated osteomyelitis and postoperative infection after orthopedic surgery. The loading capacity of Bicera™ was measured to understand its potential antibiotic adsorption volume. An antibiotic susceptibility test was also carried out to analyze the effect of Bicera™ loaded with different concentrations of vancomycin on the growth inhibition of methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin-loaded Bicera™ was implanted into rabbits with bone defects, and general gross, radiographic, and histological evaluation was undertaken at 4, 12, and 24weeks after implantation. Results: The maximum loading capacity of vancomycin-loaded Bicera™ was 0.9ml of liquid regardless of the vancomycin concentration. Antibiotic susceptibility tests showed that vancomycin-loaded Bicera™ inhibited the growth of MRSA for 6weeks. In addition, animal studies revealed that new bone grew into the vancomycin-loaded Bicera™. The percentage of new bone formation from 4 to 24weeks after implantation increased from 17% to 36%. Conclusion: Vancomycin-loaded Bicera™ could effectively inhibit the growth of MRSA in vitro. It was found to incorporate into the host bone well, and new bone was able to grow within the bone substitute. The results of this study indicate that vancomycin-loaded Bicera™ is a potential bone substitute that can prevent implant-associated osteomyelitis and postoperative infection.

Synthesis of the cyanobacterial halometabolite Chlorosphaerolactylate B and demonstration of its antimicrobial effect in vitro and in vivo.

Chlorosphaerolactylate B, a newly discovered antimicrobial halometabolite from the cyanobacterium Sphaerospermopsis sp. LEGE 00249 has been synthesized in three steps by using 12-bromododecanoic acid as starting material. A total of 0.5 g was produced for in vitro and in vivo antimicrobial efficacy testing. In vitro, the minimal inhibitory concentration (MIC) was estimated to be 256 mg/L for Staphylococcus aureus, while the minimal biofilm inhibitory concentration (MBIC) was estimated to be 74 mg/L. The in vivo study utilized a porcine model of implant-associated osteomyelitis. In total, 12 female pigs were allocated into 3 groups based on inoculum (n = 4 in each group). An implant cavity (IC) was drilled in the right tibia and followed by inoculation and insertion of a steel implant. All pigs were inoculated with 10 μL containing either: 11.79 mg synthetic Chlorosphaerolactylate B + 104 CFU of S. aureus (Group A), 104 CFU of S. aureus (Group B), or pure saline (Group C), respectively. Pigs were euthanized five days after inoculation. All Group B animals showed macroscopic and microscopic signs of bone infection and both tissue and implant harbored S. aureus bacteria (mean CFU on implants = 1.9 × 105). In contrast, S. aureus could not be isolated from animals inoculated with saline. In Group A, two animals had a low number of S. aureus (CFU = 6.7 × 101 and 3.8 × 101, respectively) on the implants, otherwise all Group A animals were similar to Group C animals. In conclusion, synthetic Chlorosphaerolactylate B holds potential to be a novel antimicrobial and antibiofilm compound.

Locally delivered antistaphylococcal lysin exebacase or CF-296 is active in methicillin-resistant Staphylococcus aureus implant-associated osteomyelitis.

Introduction: Staphylococcus aureus is the most common cause of orthopedic infections and can be challenging to treat, especially in the presence of a foreign body. The antistaphylococcal lysins exebacase and CF-296 have rapid bactericidal activity, a low propensity for resistance development, and synergize with some antibiotics. Methods: Rabbit implant-associated osteomyelitis was induced by drilling into the medial tibia followed by locally delivering exebacase, CF-296, or lysin carrier. A titanium screw colonized with methicillin-resistant S. aureus (MRSA) IDRL-6169 was inserted. Intravenous daptomycin or saline was administered and continued daily for 4 d. On day 5, rabbits were euthanized, and the tibiae and implants were collected for culture. Results were reported as log colony forming units (cfu) per gram of bone or log cfu per implant, and comparisons among the six groups were performed using the Wilcoxon rank sum test. Results: Based on implant and bone cultures, all treatments resulted in significantly lower bacterial counts than those of controls (). Exebacase alone or with daptomycin as well as CF-296 with daptomycin were more active than daptomycin alone () or CF-296 alone () based on implant cultures. CF-296 with daptomycin was more active than either CF-296 alone () or daptomycin alone () based on bone cultures. Conclusion: Local delivery of either exebacase or CF-296 offers a promising complement to conventional antibiotics in implant-associated infections.

Efficacy of Bisphosphonate-Conjugated Sitafloxacin in a Murine Model of S. aureus Osteomyelitis: Evidence of "Target & Release" Kinetics and Killing of Bacteria Within Canaliculi.

S. aureus infection of bone is difficult to eradicate due to its ability to colonize the osteocyte-lacuno-canalicular network (OLCN), rendering it resistant to standard-of-care (SOC) antibiotics. To overcome this, we proposed two bone-targeted bisphosphonate-conjugated antibiotics (BCA): bisphosphonate-conjugated sitafloxacin (BCS) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS). Initial studies demonstrated that the BCA kills S. aureus in vitro. Here we demonstrate the in vivo efficacy of BCS and HBCS versus bisphosphonate, sitafloxacin, and vancomycin in mice with implant-associated osteomyelitis. Longitudinal bioluminescent imaging (BLI) confirmed the hypothesized “target and release”-type kinetics of BCS and HBCS. Micro-CT of the infected tibiae demonstrated that HBCS significantly inhibited peri-implant osteolysis versus placebo and free sitafloxacin (p < 0.05), which was not seen with the corresponding non-antibiotic-conjugated bisphosphonate control. TRAP-stained histology confirmed that HBCS significantly reduced peri-implant osteoclast numbers versus placebo and free sitafloxacin controls (p < 0.05). To confirm S. aureus killing, we compared the morphology of S. aureus autolysis within in vitro biofilm and infected tibiae via transmission electron microscopy (TEM). Live bacteria in vitro and in vivo presented as dense cocci ~1 μm in diameter. In vitro evidence of autolysis presented remnant cell walls of dead bacteria or “ghosts” and degenerating (non-dense) bacteria. These features of autolyzed bacteria were also present among the colonizing S. aureus within OLCN of infected tibiae from placebo-, vancomycin-, and sitafloxacin-treated mice, similar to placebo. However, most of the bacteria within OLCN of infected tibiae from BCA-treated mice were less dense and contained small vacuoles and holes >100 nm. Histomorphometry of the bacteria within the OLCN demonstrated that BCA significantly increased their diameter versus placebo and free antibiotic controls (p < 0.05). As these abnormal features are consistent with antibiotic-induced vacuolization, bacterial swelling, and necrotic phenotype, we interpret these findings to be the initial evidence of BCA-induced killing of S. aureus within the OLCN of infected bone. Collectively, these results support the bone targeting strategy of BCA to overcome the biodistribution limits of SOC antibiotics and warrant future studies to confirm the novel TEM phenotypes of bacteria within OLCN of S. aureus-infected bone of animals treated with BCS and HBCS.

Delineating the Role of the msaABCR Operon in Staphylococcal Overflow Metabolism

Staphylococcus aureus is an important human pathogen that can infect almost every organ system, resulting in a high incidence of morbidity and mortality. The msaABCR operon is an important regulator of several staphylococcal phenotypes, including biofilm development, cell wall crosslinking, antibiotic resistance, oxidative stress, and acute and chronic implant-associated osteomyelitis. Our previous study showed that, by modulating murein hydrolase activity, the msaABCR operon negatively regulates the proteases that govern cell death. Here, we report further elucidation of the mechanism of cell death, which is regulated by the msaABCR operon at the molecular level in the USA300 LAC strain. We showed that deletion of msaABCR enhances weak-acid-dependent cell death, because, in the biofilm microenvironment, this mutant strain consumes glucose and produces acetate and acetoin at higher rates than wild-type USA300 LAC strain. We proposed the increased intracellular acidification leads to increased cell death. MsaB, a dual-function transcription factor and RNA chaperone, is a negative regulator of the cidR regulon, which has been shown to play an important role in overflow metabolism and programmed cell death during biofilm development in S. aureus. We found that MsaB binds directly to the cidR promoter, which represses expression of the cidR regulon and prevents transcription of the cidABC and alsSD operons. In addition, we observed that pyruvate induced expression of the msaABCR operon (MsaB). The results reported here have enabled us to decipher the role of the msaABCR operon in staphylococcal metabolic adaption during biofilm development.

Rifaximine spacer application is not superior to local teicoplanin treatment in a rat model of osteomyelitis.

Acute and chronic osteomyelitis generally require long-term antibiotic therapy and surgical debridement. Implant-associated osteomyelitis, particularly from methicillin-resistant Staphylococcus aureus (MRSA) strains, is difficult to treat. Rifaximin is an antibiotic derived from rifamycin which may be effective in the treatment of osteomyelitis in terms of its wide spectrum of action and pharmacological properties. The aim of this experimental study was to investigate the local efficacy of rifaximin in rat models with MRSA and implant associated osteomyelitis. This study was carried out with 40 adult Wistar albino rats. The rats were randomly divided into 4 equal groups with 10 rats in each. An implant related MRSA osteomyelitis was created in the right tibia metaphysis of each rat by Norden's experimental osteomyelitis model. After 4 weeks, the implants of each tibia were removed and debridement was applied. Group 1 was designed as control group and no other treatment was applied other than debridement. Bone cement without any antibiotic was applied to Group 2, bone cement with teicoplanin was applied to Group 3 and bone cement with rifaximin was applied to Group 4. After 4 weeks from the second surgery, euthanasia was performed to the rats and the clinical, histopathological and microbiological results were compared. There was no statistically significant difference between the groups in clinical scoring. A statistically significant difference was found between the histopathological scores of Group 1 and Group 2 and the histopathological scores of Groups 3 and 4; the histopathological scores of Group 1 and Group 2 were found to be higher than Group 3 and Group 4. When the pre-and post-treatment colony numbers were compared, although there was a statistically significant difference between Group 3 and Group 2, no statistically significant difference was found between Group 4 and Group 1 results. In spite of its wide spectrum, the local efficacy of rifaximin in the treatment of osteomyelitis could not be demonstrated. This study shows the ability to shed light on some future comprehensive studies with the inclusion of infection markers.

Terminology of bone and joint infection.

Bone & Joint ResearchVol. 10, No. 11 InfographicOpen AccessOpen Access licenseTerminology of bone and joint infectionCrossmarkMarkus Rupp, Nike Walter, Susanne Baertl, Siegmund Lang, David W. Lowenberg, Volker AltMarkus RuppConsultantMarkus Rupp. E-mail: E-mail Address: [email protected]https://orcid.org/0000-0001-7221-3783 Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany Search for more papers by this author, Nike WalterResearch Associatehttps://orcid.org/0000-0002-0654-2076 Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany Search for more papers by this author, Susanne BaertlResident Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany Search for more papers by this author, Siegmund LangResident Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany Search for more papers by this author, David W. LowenbergClinical Professor Department for Orthopaedic Surgery, Stanford Medical School, Redwood City, California, USA Search for more papers by this author, Volker AltDirector and Chairmanhttps://orcid.org/0000-0003-0208-4650 Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany Search for more papers by this authorPublished Online:17 Nov 2021https://doi.org/10.1302/2046-3758.1011.BJR-2021-0371AboutSectionsPDF/EPUB ToolsDownload CitationsTrack CitationsAdd to Favourites ShareShare onFacebookTwitterLinked InRedditEmail Musculoskeletal infections are common and often difficult to treat.1,2 Their diagnostics and treatment are an integral part of the daily clinical routine of every orthopaedic and trauma surgeon. Diagnostics and first treatment approaches are often initiated prior to referrals to a specialized centre. Treatment algorithms in the field of musculoskeletal infections, based on clinical and experimental empiricism, promise successful treatment courses in many cases,3-5 although a considerable proportion of bone and joint infections are characterized by treatment failure, for a variety of reasons.6The basic prerequisite for the initiation of correct diagnostics, and thus consecutive best possible therapy, is the correct terminology of individual bone and joint infections. A variety of terms exists to describe bone and joint infections. Osteomyelitis, implant-associated infection, fracture-related infection (FRI), and infected nonunion denote bone infections. Joint infections are described by periprosthetic joint infection (PJI) and septic arthritis, with its synonyms of supportive and infectious arthritis. In the spine, a distinction can be made between implant-associated vertebral osteomyelitis and hematogenous spondylodiscitis according to the aetiology. Even among experts in the field, terms are interchanged and used synonymously. It often appears to be difficult to arrive at exact definitions, which naturally limits the understanding of optimal treatment. For FRI and PJI, diagnostic criteria have been developed, and in the case of PJI those criteria have been continuously improved and are still the subject of scientific discussion.7-9To lay the foundation for optimal therapy using the correct terminology of the underlying condition, this infographic presents the hallmarks of bone and joint infections and diagnostic criteria.