Eur J Clin Microbiol Infect Dis (2013) 32:195–198 DOI 10.1007/s10096-012-1731-6
ARTICLE
A delivery system of linezolid to enhance the MRSA osteomyelitis prognosis: in vivo experimental assessment A. Gaudin & C. Jacqueline & H. Gautier & C. Desessard & V. Le Mabecque & A.-F. Miegeville & G. Potel & J.-M. Bouler & P. Weiss & J. Caillon & G. Amador
Received: 7 May 2012 / Accepted: 13 August 2012 / Published online: 25 August 2012 # Springer-Verlag 2012
Abstract Staphylococcus aureus, a major responsible microorganism of osteomyelitis, represents a challenge to treat because of the poor penetration of antibiotics in bone and increasing minimum inhibitory concentrations (MICs) to glycopeptides. The calcium-deficient apatites (CDA), closer to the biological components found in bone and other calcified tissues, have osteoconductive properties. So, to process severe osseous infections, CDA can be used to deliver in the infectious site antibiotics like linezolid. The acute experimental osteomyelitis due to methicillin-resistant Staphylococcus aureus (MRSA) was induced in rabbit’s femurs and surgery mimicking human procedures was performed at day three after inoculation. Animals were randomly assigned to treatment groups: L(IV) [4-day linezolid IV infusion, human-equivalent dose of 10 mg/kg/12 h], L(CDA50%) (100 mg CDA with linezolid 500 μg/mg) and L(CDA50%) +L(IV). Surviving bacteria were counted in bone marrow (BM) and bone (Bo) at day 3 (before treatment), day 7 (4-day treatment) or day 17 (14-day treatment). L(iv) was effective after a 4-day treatment with a log10CFU/g decrease of −2.63±1.92 and −2.17±1.58 in bone marrow A. Gaudin : C. Jacqueline : C. Desessard : V. Le Mabecque : A.-F. Miegeville : G. Potel : J. Caillon : G. Amador (*) Faculté de Médecine, UPRES EA 3826, Université de Nantes, 1 rue Gaston Veil, Nantes 44000, France e-mail:
[email protected] H. Gautier : J.-M. Bouler : P. Weiss INSERM UMRS 791, Laboratory of Osteoarticular and Dental Tissue Engineering, 1 place Alexis Ricordeau, Nantes 44000, France C. Jacqueline : G. Potel : J. Caillon : G. Amador CHU Nantes, 1 place Alexis Ricordeau, Nantes 44000, France
and bone, respectively. CDA loaded with linezolid enhance the efficacy of the IV linezolid regimen by more than one log10CFU/g.
Introduction Osteomyelitis and osteitis represent the majority of severe bone infections, and are caused by a microorganism in the bloodstream that gains access to bone tissue. Staphylococcus aureus is isolated in 60 % of bone infection cases [9] and, of these isolates, the methicillin resistance rate is increasing [7]. Staphylococcus aureus bone infection causes rapid bone destruction [6] and spread of the infection to surrounding tissues, making these infections responsible for significant morbidity and healthcare costs each year. The treatment of sub-acute or chronic forms of osteomyelitis is complex and requires 6–12 months of treatment, as well as multiple surgical procedures to remove intra-osseous or subperiostium abscesses and sequestrum bone [3]. Vancomycin is the reference antibiotic against methicillin-resistant Staphylococcus aureus (MRSA) strains [8] but it requires the monitoring of serum drug levels and adverse effects are not negligible. Further, poor penetration of these drugs into osseous tissues is likely the reason for treatment failures or recurrence of the infection [2] that underlines the need for new therapeutic alternatives. Unfortunately, therapeutic options for bone infections are extremely limited, so new approaches are needed in order to overcome the increasing resistance of Staphylococcus aureus strains to vancomycin and to improve the efficiency of antimicrobial therapy for bone and joint infections. When the minimum inhibitory concentration (MIC) of a Staphylococcus aureus isolate to vancomycin is greater than
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2 μg/mL, linezolid, an antibiotic inhibiting the protein synthesis, represents the most consensual alternative. This drug is generally reserved for the treatment of severe infections in soft and skin tissues, but it could have great potential benefits for the treatment of bone and joint infections. However, because clinical trials for the antibiotic treatment of osteomyelitis are rare and difficult to perform, animal studies are the most appropriate and feasible way to assess the impact of antibiotic therapy on the outcome of bone infections. Administering antibiotics locally rather than systemically could decrease the emergence of resistant mutants due to a more direct effect at the site of infection. This could also limit or prevent the occurrence of side effects, such as drug toxicity. Rauschmann et al. [10] studied in vitro a nanocrystalline apatite (PerOssal®) as a delivery system for antibiotics and demonstrated that this material was able to release an active molecule (vancomycin and gentamicin) during a 10-day period without causing drug toxicity. In this study, we evaluated whether calcium-deficient apatites (CDA) could be used as a local delivery system (LDS) for linezolid [5]. The objective of our work was to enhance the efficacy of the antimicrobial systemic treatment by bringing a high local dose of antibiotic and then accelerate the healing process due to the availability of calcium and phosphate ions from the CDA.
Materials and methods Animals Female New Zealand rabbits (weight 2.0 to 2.5 kg) were used in this study. The rabbits were individually caged and had free access to water and food. Experiments were performed according to the Committee of Animal Ethics of the University of Nantes, France. Moribund animals (i.e. having difficulty accessing water and food associated with 10 % weight loss per day for 2 days) were euthanised by lethal injection of thiopental under general anaesthesia. Bacterial strain The MRSA strain used in this study was isolated from a blood culture and exhibited heterogeneous low-level methicillin resistance (methicillin MIC016 μg/mL). The MRSA MIC was 2 μg/mL for linezolid. LDS synthesis Synthesis of granules containing 50 % linezolid weight/ weight was realised in the INSERM laboratory by wet
Eur J Clin Microbiol Infect Dis (2013) 32:195–198
granulation [5]. Nuclear magnetic resonance spectroscopy showed no change in the structure of each antibiotic when linked to CDA. These results were confirmed by studying the antibacterial activity of antibiotics, which remained proportional to the quantity of loaded molecules, proving that the released compound was active. The in vitro release time of the CDA granules containing 50 % linezolid was 26 days (20 % released in the first 4 days and 50 % released in the first 10 days). The size of the granules was determined to be 200–500 μm, compatible with a common handling human practice.
Experimental design We used a percutaneously transarticular route to perform a femoral trepanation using a Jamshidi bone marrow biopsy needle (8 Ga) under general anaesthesia (ketamine 20 mg/kg IV, and xylazine, 1 mg/kg IV) [4]. A fentanyl patch (Durogesic®, Janssen-Cilag Lab.) was used for pain management during the study. The Jamshidi needle was inserted between the two femoral condyles and through the epiphysis, physis and metaphysis to reach the medullary canal. After the needle was removed, the skin incision was closed. A 1-mL suspension containing 109 CFU MRSA was injected into the knee cavity. By day 3 post inoculation, the animals have acute MRSA osteomyelitis. This was treated by debridement of the site of infection, as is recommended for humans [9]. Samples of bone marrow (BM) and spongy bone (Bo) were obtained for bacterial counts. The antibacterial activity of this LDS was tested in the absence or presence of a standard systemic antibiotic treatment using an in vivo model of acute MRSA osteomyelitis [4]. Local antibacterial activity was assessed after 4 days and 14 days of linezolid LDS osseo implantation. Animals were randomly assigned to three different treatment groups: (1) L(IV): IV linezolid using a computer-controlled syringe pump infusion simulating a human-equivalent dose of 10 mg/kg/12 h; (2) L(CDA50%): CDA loaded with 500 μg/mg of linezolid alone; (3) L(CDA50%) +L(IV): CDA loaded with 500 μg/mg of linezolid in addition to the IV linezolid infusion. The infusion of linezolid started on day 3 and continued only for 4 days for ethical reasons. Each group was assessed after 4 days and 14 days of local treatment, except for the L(IV) group. At day 7 or day 17 (corresponding to a 4-day or 14day LDS treatment, respectively), the animals were euthanised using a 100-mg IV bolus of thiopental, and samples of BM and bone were taken to measure the bacterial load. The results were expressed as the difference in the bacterial counts (log10CFU/g of tissue) between day 3 (before treatment) and at the end of the treatment, day 7 or day 17. The lower limit of detection for this method was 1 CFU/50 μl of undiluted tissue homogenate.
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Bacterial counts
Discussion
Bacterial counts were determined after 48 h of incubation at 37 °C on Tryptic soy agar plates. To evaluate the linezolid treatment-induced selection of in vivo resistant variants, undiluted sample homogenates were spread on agar plates containing 8 μg/mL linezolid, 4-fold greater than the MIC.
This acute model of osteomyelitis allowed to study early stages of osseous infection. The failures of the antibiotics regimens are frequent and bacteria invasion of bone tissues leading to bone sequestrum, biofilm or micro-colonies is still difficult to treat. A filling biomaterial, used in addition to the usual surgical and antibiotic treatments, could enhance the prognosis of these severe infections, needing most often several repeated surgical procedures and multiple antibiotherapies. First, the 50 % linezolid-loaded CDA exhibit the same efficacy as systemically administered linezolid alone. A trend to lower bacterial counts was observed, but this trend did not reach statistical significance. This could be due to the number of concerned animals, limiting the power of the statistical test. Further, none of the tissue samples were sterile after the 14-day treatment. Our data suggest that the use of an LDS should be combined with at least a short-term systemic antibiotic treatment in the first several days after surgery. In human therapeutics, it would be very interesting to associate our linezolid carrier able to deliver linezolid in a local way in high concentration to a prolonged biotherapy including linezolid, allowing the non-emergence of mutant bacteria. Prolonged antibiotic regimens cannot be assessed in animal models because of major venous impairment due to the local toxicity of the molecule. Nevertheless, the results at 14 days showed a marked decrease in the bacterial counts in bone and bone marrow, a decrease which cannot be due to the infusion of linezolid alone or the immune system of rabbits, our model being stable over the study period. While more traditional antibiotic carrier systems are available, CDA have both desirable antibiotic release kinetics and high osteogenic-promoting activity, including degradation of the apatite, obviating the need for a second surgery to remove the implanted material. Due to the similarity between bone tissue and CDA, this matrix appears to be an interesting prospect for an LDS. It can be used to accelerate the healing process in operative sites due to its intrinsic osteoconductive properties [1]. The principal clinical challenge for the treatment of bone infections is sterilising the bone tissues. By combining CDA loaded with 50 % linezolid and an early IV or IM antibiotic
Statistical analysis Statistical analyses were performed using GraphPad Prism® 4 for Windows (GraphPad Software, San Diego, CA, USA). The results were expressed as means ± standard deviation. Regimens were compared using one-way analysis of variance. This analysis was performed using a post hoc Student-Newman–Keuls test. Time-dependant efficacy was tested by a non-parametric t-test associated to a Wilcoxon post-test. A p-value< 0.05 was considered to be statistically significant.
Results The mean bacterial counts on day 3 post inoculation in BM and Bo were 7.86 ± 0.55 and 7.53 ± 0.69 CFU/g of tissue, respectively. Each rabbit was its own control for bacterial counts. IV infusion of linezolid (L(IV)) showed significant antibacterial activity in both BM and Bo after 4 days of treatment. L(CDA50%) alone and combined with L(IV) enhanced the bacterial reduction, but it was not statistically significant. After 14 days of treatment, the bacterial reduction increased in bone marrow with L(CDA50%) combined with L(IV) (Table 1). L(CDA50%) alone did not exhibit greater efficacy after the 14-day treatment than after the 4-day treatment. The infusion of linezolid was important to improve the efficacy of the LDS for bone marrow. No linezolid-resistant mutants were detected after 4 or 14 days of treatment in any treatment group or in any compartment.
Table 1 Efficacy of antibiotic regimens on bacterial counts in bone marrow and bone after a 4-day treatment Treatment regimens
n
Mean±SD Δlog10 CFU/g of tissue (day 7–day 3) Bone marrow
L(IV) L(CDA50%) L(CDA50%) +L(IV)
8 7 7
n number of animals; ND not done
−2.63±1.92 −3.10±0.84 −3.67±0.65
n
Bone −2.17±1.58 −1.73±0.94 −3.02±0.60
Mean±SD Δlog10 CFU/g of tissue (day 17–day 3) Bone marrow
5 10
ND −2.98±1.08 −4.24±1.90
Bone ND −1.80±1.89 −3.11±2.23
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treatment, CDA loaded with linezolid could enhance the efficacy of the IV linezolid regimen and may reduce the morbidity and the social cost of this type of severe bone infection by limiting the duration of treatment in human practice.
Conflict of interest The authors declare that they have no conflict of interest.
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