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Efficacy of intramuscular moxifloxacin in the treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus
Accepted Manuscript Title: Efficacy of intramuscular moxifloxacin in the treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus Author: Vasileios Soranoglou, Ilias Galanopoulos, Evangelos J. GiamarellosBourboulis, Apostolos Papalois, Efthymia Giannitsioti, Lazaros A. Poultsides, Theodosia Choreftaki, Kyriaki Kanellakopoulou PII: DOI: Reference:
S0924-8579(17)30175-9 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.041 ANTAGE 5121
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
4-10-2016 28-1-2017
Please cite this article as: Vasileios Soranoglou, Ilias Galanopoulos, Evangelos J. GiamarellosBourboulis, Apostolos Papalois, Efthymia Giannitsioti, Lazaros A. Poultsides, Theodosia Choreftaki, Kyriaki Kanellakopoulou, Efficacy of intramuscular moxifloxacin in the treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Efficacy of intramuscular moxifloxacin in the treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus
Vasileios Soranoglou a,*, Ilias Galanopoulos b, Evangelos J. Giamarellos-Bourboulis c, Apostolos Papalois d, Efthymia Giannitsioti c, Lazaros A. Poultsides e, Theodosia Choreftaki f, Kyriaki Kanellakopoulou c
a
2nd Department of Orthopaedics, Athens General Hospital, Athens, Greece
b
1st Department of Orthopaedics, General Military Hospital, Athens, Greece
c
4th Department of Internal Medicine, University of Athens, Medical School, Athens, Greece
d
ELPEN Pharmaceutical Company, Pikermi, Greece
e
Adult Reconstruction and Joint Replacement Division, Department of Orthopaedic Surgery,
Hospital for Special Surgery, New York, USA f
Department of Pathology, Athens General Hospital, Athens, Greece
*
Corresponding author. Present address: Adult Reconstruction and Joint Replacement
Division, Department of Orthopaedic Surgery, Hospital for Special Surgery, 535 East 70th St, 10021, New York, USA. E-mail address: [email protected] (V. Soranoglou).
ARTICLE INFO Received 4 October 2016 Accepted 28 January 2017
Keywords: 1 Page 1 of 23
Moxifloxacin Osteomyelitis treatment Intramuscular Bone concentration MRSA
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1
Highlights
2
osteomyelitis model.
3 4
Intramuscular moxifloxacin treatment proved effective in an experimental
Moxifloxacin bone levels were approximately 43 times higher than the minimum
5
inhibitory
6
Staphylococcus aureus.
7 8
concentration
for
the
tested
strain
of
methicillin-resistant
No differences in moxifloxacin bone levels were found between healthy and infected tibias.
9 ABSTRACT Fluoroquinolones have been well studied in the treatment of chronic osteomyelitis due to their beneficial pharmacokinetic (PK) and pharmacodynamic profiles. The purpose of this study was to determine the efficacy of intramuscular (IM) moxifloxacin administration in the treatment of experimental osteomyelitis by methicillin-resistant Staphylococcus aureus.
Following an experimental osteomyelitis animal model described previously, three groups of rabbits (A=control; B=IM moxifloxacin administration; C=PK study of moxifloxacin penetration into bone) were evaluated. Three weeks after bacterial inoculation, surgical debridement was performed in all animals and IM treatment commenced for Groups B and C. Sacrifice was performed in an A:B group animal ratio of 1:2 at weekly intervals from 7th to 42nd day post debridement and from 21st to 56th day post debridement for Groups A and B, respectively (including 2-week interval without antibiotics for Group B). Cancellous bone was harvested for microbiological and histopathological analyses at re-operation and sacrifice for Groups A and B. Cortical bone moxifloxacin levels were measured in Group C following 7, 14, 35 and 42 days of treatment.
In Group A, bacterial growth after surgical debridement was significant, whereas high eradication rates were observed in Group B. Radiological abnormalities and histopathological 3 Page 3 of 23
findings were evaluated. Moxifloxacin bone levels, observed in Group C, were approximately 43 times greater than the minimum inhibitory concentration, with no difference found between infected and healthy tibial bone.
The therapeutic protocol was very effective in this model of experimental osteomyelitis. However, further evaluation of these results in clinical studies is crucial. 1. Introduction Chronic osteomyelitis is one of the most difficult infections to treat in humans [1]. Treatment requires accurate identification of the causative micro-organism and sensitivity to the antimicrobial agents. Classical documented therapeutic options include intravenous (IV) administration of antimicrobial agents for at least 4–6 weeks along with operative intervention. The latter may include radical surgical debridement, elimination of dead space, bone and soft tissue reconstruction, and skin coverage with grafts [1–6]. However, despite the well-established treatment protocols, significant recurrence rates are still reported in the literature [7]. Inability to achieve high local antimicrobial concentrations in the infected bone and surrounding soft tissue area following systemic administration is one of the factors that may compromise the definitive treatment of chronic osteomyelitis.
Fluoroquinolones, particularly ciprofloxacin, are used in the treatment of osteomyelitis due to their excellent activity against most common pathogens and low systemic toxicity. Furthermore, they show a favourable pharmacokinetic (PK) profile, consisting of high bioavailability (60–99%), moderate protein binding (20–60%) and good tissue penetration [8]. Additionally, their ability to penetrate into cells may be advantageous in bone infections, as in-vitro studies have shown that Staphylococcus aureus can penetrate into and survive in osteoblasts [9].
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Moxifloxacin, a fourth-generation fluoroquinolone, appears to be a promising, clinically safe agent with beneficial features in the era of bone infections [9–13]. Its long elimination halflife of 13 h means that it can be dosed daily, whereas other antimicrobial agents (e.g. ciprofloxacin, vancomycin, clindamycin) require more frequent administration. A systematic review of antibiotic penetration into bone [9] showed higher bone:serum concentration ratios for moxifloxacin (mean range 0.33–1.05) compared with glycopeptides (mean range 0.1–0.6), clindamycin (mean range 0.21–0.45) and rifampicin (mean range 0.08–0.57). Additionally, moxifloxacin demonstrates excellent bioavailability after oral administration [13], comparable with that of clindamycin [14]. This is extremely valuable when considering the long duration of systemic therapeutic regimens for osteomyelitis that usually require central venous catheters for parenteral administration. Appropriately chosen oral agents demonstrate clinical equivalence compared with parenteral osteomyelitis therapy [15].
This study evaluated an intramuscular (IM) moxifloxacin therapeutic protocol in the setting of an established experimental model of methicillin-resistant S. aureus (MRSA) osteomyelitis. The aim of this study was to assess: (i) cure or eradication rates of the infection by parenteral treatment; and (ii) evaluation of bone moxifloxacin levels in systemically treated subjects.
2. Materials and methods 2.1. Study design One MRSA experimental osteomyelitis model, modified from that described previously by Norden and Andriole [16–20], was utilized. Three study groups were formed: A (control, n=18), B (IM moxifloxacin therapy, n=36) and C (PK study of IM moxifloxacin bone
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penetration, n=12). To avoid sacrifice of an extreme number of animals, it was considered that PK studies including animals at a 1:3 ratio of the treatment group would be sufficient.
2.2. Animal model In total, 66 white New Zealand male rabbits (body weight 2.3–3.6 kg) were included in the study. Animals were housed in single metal cages and had access to tap water and standard balanced rabbit chow ad libitum. Room temperature ranged between 18 ◦C and 22 ◦C, relative humidity ranged between 55% and 65%, and the light/dark cycle was 06:00/18:00 h.
2.3. Pathogen The MRSA isolate was derived from a patient who presented with chronic osteomyelitis. The minimum inhibitory concentration (MIC) of moxifloxacin was determined by a microdilution technique in 96-well plates using a 5x105 colony-forming units (cfu)/mL log-phase inoculum at a final volume of 0.1 mL in Mueller–Hinton broth (Becton-Dickinson, Franklin Lakes, NJ, USA); this was ≤0.015 μg/mL. After growth for 5 h at 37 ◦C in a shaking water bath in Mueller–Hinton broth, a 1×108 cfu/mL inoculum was prepared and applied for bacterial challenge, as described below.
2.4. Antibacterial agent Pure moxifloxacin hydrochloride powder (Bayer Healthcare AG, Berlin, Germany) was admixed with sterile water for injections BP (B. Braun Melsungen AG, Melsungen, Germany) and vortex stirred to a sterile 2% aqueous solution (20 mg/mL) which was used for the IM injections. The solution was prepared daily. Unused solution and solutions with visible undissolved particles were discarded. The selected dose was 5 mg/kg body weight once per day based on the study of Fernández-Varón et al. [21].
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2.5. Experimental animal model assay Osteomyelitis was induced surgically in the right upper tibial metaphysis in all rabbits. Animals were sedated by IM injection of 25 mg/kg body weight ketamine 10% (Νarketan 10, Vetoquinol AG, Belp-Bern, Switzerland) and 5 mg/kg body weight xylazine 2% (Rompun Bayer AG, Leverkusen, Germany). Anaesthesia was maintained by IM administration of 15 mg/kg body weight xylazine at 30-min intervals. A 2-cm skin incision was made 2.5 cm distal to the knee joint, exposing the anteromedial cortex of the proximal tibial metaphysis. Using a 2-mm drill, a hole was made and 0.1 mL of the prepared bacterial suspension was instilled. A 25G needle was placed through the hole, serving as a foreign body. The needle was curved at one end in order to be fixed securely on to the tibial cortex. Finally, the hole was plugged with sterile bone wax.
The infection was allowed to develop for 3 weeks, at which time all animals underwent a second operation for removal of the needle and debridement of necrotic and infected tissue. Cancellous bone samples were also obtained from the site of infection for quantitative cultures, and the hole was plugged with sterile bone wax. Animals were then assigned at random into three groups: Group A (n=18), without any subsequent antimicrobial treatment (control group); Group B (n=36), with initiation of IM moxifloxacin therapy of 5 mg/kg body weight dose q.d.; and Group C (n=12), which received the same treatment as Group B and served as the PK study group. Postoperative analgesia was achieved by IM paracetamol (Apotel, Uni-Ρharma, Pharmaceutical Laboratories S.A., Kifissia, Greece) administration according to standard practice for experimental model assays.
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During the experimental study, animal weight and temperature were assessed weekly and surgical trauma was inspected daily. Three animals in Group A were sacrificed at regular time intervals (i.e. 7, 14, 21, 28, 35 and 42 days after the day of reoperation) by intracardiac pentobarbital sodium 20% (Dolethal, Vetoquinol SA, Lure Cedex, France) injection after sedation. Animals in Group B were treated for periods of 7, 14, 21, 28, 35 and 42 days (six animals in each treatment period), and were sacrificed following a 2-week interval without antibiotics. The rationale behind this interval was to evaluate any potential regrowth of S. aureus bone infection [6]. On sacrifice, infected tibias were harvested and specimens of cancellous bone were obtained under sterile conditions for quantitative culture and biopsy. Animals in Group C were divided into four equal subgroups and received IM treatment for 7, 14, 35 and 42 days, respectively, before sacrifice. Cortical bone samples were obtained from the site of infection and the corresponding site of the healthy tibia 24 h after the last IM dose, and used for measurement of moxifloxacin bone levels.
2.6. Bone cultures Cancellous bone samples harvested during re-operation and upon sacrifice were weighed and homogenized by a sterile tissue grinder. Samples were suspended in 1 mL of sterile saline solution and cultured quantitatively by seven consecutive 1:10 dilutions in sterile water. A 0.1-mL aliquot of each dilution was then plated on to McConkey agar (Becton Dickinson, Cockeysville, MD, USA). This serial subculturing allowed avoidance of any antibiotic carryover effect. Bacterial growth was expressed as cfu/g tissue. The lowest detection limit was 10 cfu/g.
2.7. Histopathology Separate bone particles harvested on sacrifice from Group A and B animals were fixed in neutral buffered formalin, embedded in paraffin wax, sectioned and stained with 8 Page 8 of 23
haematoxylin and eosin. A semiquantitative scoring system, adopted with modifications from previous studies [20,22], was used to grade acute and chronic inflammation, and giant cell infiltration. The first two parameters were scored as 0 (absent), 1 (sparse), 2 (moderate) and 3 (intense). Giant cell infiltration was graded as 1 (positive) or 0 (negative). As all histological parameters were given equal diagnostic weight, a composite (arithmetic sum) score was calculated for each animal and used for the comparative evaluation.
2.8. Radiology Plain anteroposterior and lateral radiographs of the infected tibia were taken twice for each animal in Groups A and B using exposure factors of 36 keV and 25 ms. The first radiograph was taken just before re-operation and the second radiograph was taken at the completion of treatment just before animal sacrifice. During x-rays, animals were sedated and small sandbags were used to immobilize the leg in the proper position.
2.9. PK study of moxifloxacin bone penetration The grinding procedure was repeated for each sample of cortical bone collected from the two corresponding sites in Group C. Adopting a technique described previously [20], samples were centrifuged for 10 min at 1400×g at 4 ◦C, after suspension of 1 mL of sterile sodium saline. Supernatants were collected and stored at −80 ◦C. Moxifloxacin concentrations were estimated in bone supernatants by high-performance liquid chromatography (HPLC). Briefly, 250 μL of sample was diluted with 250 μL of displacing reagent and centrifuged for 5 min at 2700×g. The displacing reagent consisted of 0.5% sodium dodecyl sulphate (SDS) (Merck, Darmstadt, Germany) and 20% acetonitrile (Merck) in 75mM sodium phosphate buffer (pH 7.5). Ten microlitres of the supernatant were injected into an HPLC system (Agilent 1100 Series; Agilent Technologies, Waldbronn, Germany) with the following elution
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characteristics: Nucleosil 100-5 C18 (4.6 mm×250 mm, 5 μm) column at 37 ◦C; mobile phase consisting of a pH 3.0 buffer composed of 25 mM Na3PO4 and 10 mM SDS and acetonitrile 99% at a 35/65 ratio with a flow rate of 1 mL/min; and ultraviolet detection at 293 nm. The retention time of moxifloxacin was 2.9 min and its concentration was estimated (in μg/mL) using a standard curve ranging between 0.095 and 100 μg/mL. All determinations were performed in duplicate. R2 of the curve was 0.9997. The interday variation of the assay was 4% [20].
2.10. Statistical analysis Bacterial growth and histological scores were expressed as mean ± standard error, and drug levels were expressed as median and interquartile range. Comparisons between groups were performed using analysis of variance with post-hoc Bonferroni corrections. Statistical significance was set at P<0.05.
3. Results The mean log10 ± standard deviation of bacterial growth in cancellous bone of all tested animals upon re-operation before assignment into each of the three groups (A, B, and C) was 4.44±1.38 cfu/g. Bacterial growth from cancellous bone of Group B animals was below the detection limit after 28 and 42 days of treatment, and was lower than the respective growth in Group A at all days of sacrifice. This difference between Group A and Group B upon sacrifice was significant at 14 days (P=0.015), 21 days (P=0.011), 28 days (P=0.006) and 42 days (P=0.006) (Fig. 1). After ≥28 days of treatment, bacterial regrowth was only documented in one of 18 sacrificed animals in Group B. Eradication rates were ≥67% on all sacrifice days in Group B, whereas negative culture results upon sacrifice were only documented in one animal in Group A.
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Clinical examination of infected animals before their second operation revealed various degrees of localized or diffuse limb inflammation. Increased local temperature, abscesses and pus draining fistulas were the main macroscopic findings. The clinical image was significantly improved, as expected, after osteomyelitis treatment in Group B. Radiographic signs of osteomyelitis were observed 3 weeks after infection. These included periosteal elevation, local demineralization and cortical irregularity with radiolucent areas. At sacrifice, radiographs in Group A revealed intense radiologic abnormalities and formation of sequestra in four animals. However, in Group B, the radiologic abnormalities were less obvious, with cortical thickening and sclerosis being the leading signs.
Comparative evaluation of the histopathological composite scores between Groups A and B revealed lower composite scores for Group B for the first 5 weeks of treatment (Fig. 2). However, differences only reached statistical significance on days 7 and 21 of sacrifice. In Group A, the grading of acute infection outweighed chronic infection for the first 3 weeks, whereas chronic infection was prevalent in Group B for the same period. Bone concentrations of moxifloxacin in cortical bone samples from infected and healthy tibias of Group C are shown in Table 1. Interestingly, almost identical drug levels were measured in healthy and infected bone samples.
4. Discussion
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The use of fluoroquinolones in the treatment of chronic osteomyelitis has been well documented over the last two decades. Parenteral fluoroquinolone administration along with surgical debridement and bone reconstruction remains an excellent treatment protocol for management. Among the fluoroquinolones, moxifloxacin shows excellent bioavailability, a long half-life and superior bone penetration [9]. Multiple in vitro and in vivo studies have shown the efficacy of moxifloxacin against Gram-positive pathogens. These studies demonstrated superiority against Staphylococcus spp. compared with older-generation fluoroquinolones, such as ciprofloxacin [23]. Moreover, the risk of resistance development during monotherapy is theoretically lower for moxifloxacin [24]. However, in the clinical setting, there are few reports for treatment of bacterial osteomyelitis and periprosthetic infection with this antibiotic [24–26].
To the best of the authors’ knowledge, no published data exist regarding the efficacy of this antimicrobial agent in IM treatment of osteomyelitis. The rationale behind using the IM route rather than the IV route was based on the fact that IV drug administration in rabbits is extremely challenging due to the lack of available veins, and it is not possible to maintain an intravenous catheter for a long time in a vigilant rabbit. Moreover, IM administration of antibiotics can result in peak serum concentrations within minutes at levels comparable to those observed after IV injections [22,23].
The study results showed that this therapeutic protocol was highly effective. After ≥4 weeks of treatment, only one of 18 animals had persistent infection. Eradication rates were ≥67% after just 1 week of treatment. A noteworthy aspect of this study was the 2-week interval between end of treatment and sacrifice. This strategy, uncommon in the literature, provided the opportunity to evaluate potential recurrence of infection. These findings should guide
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future clinical studies for further evaluation of systemic moxifloxacin administration in osteomyelitis. Furthermore, the simple dosing protocol (once per day) and safety profile of this antibiotic could overcome patient compliance issues.
A low dose (5 mg/kg body weight) of moxifloxacin was used in this study because of the very low MIC of the MRSA strain. A higher dose would possibly achieve even higher eradication rates in earlier time points. Fernández-Varón et al. [21] described moxifloxacin PK after IV, IM and oral administration in rabbits and concluded that a 5 mg/kg body weight dose would be effective against bacterial isolates with MIC ≤0.06 μg/mL. For an MIC of 0.06 μg/mL, the 24-h area under the concentration-time curve/MIC ratio was 97 h (ΙΜ). The maximum serum concentration (Cmax)/MIC ratio was approximately 10. Typically, a Cmax/MIC ratio of 8–10 has been associated with effective treatment of Gram-negative and -positive bacterial infections and prevention of resistance [27]. In this study, the MIC was ≤0.015 μg/mL.
This study also found that trough bone concentrations of moxifloxacin following IM administration were approximately 43 times higher than the MIC of moxifloxacin for the specific pathogen. However, the bacterial isolate used in this study had a very low MIC compared with strains in clinical practice, where MIC90 values (MIC for 90% of the organisms) of moxifloxacin can be up to 8 μg/mL, especially for hospital-acquired infections [27–29]. A much higher IM dose would be necessary in order to achieve therapeutic bone concentrations, but the required solution volume would be inappropriate for IM administration in rabbits.
Another important therapeutic consideration is the effect of inflammation on the penetration of antibiotic into bone. In osteomyelitis, blood flow into bone may be increased because of
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reactive hyperaemia in the infected area, or decreased due to the presence of pus and sequestra. Most of the bone penetration studies have been conducted in patients with uninfected bone [9]. In a study of osteomyelitis in rabbits, Mader et al. [30] found bone/serum concentration ratios of 0.98 for infected bone and 0.40 for uninfected bone after an IV dose of clindamycin 70 mg/kg body weight. No differences were documented in the present PK study. This is in accordance with the results of Beckmann et al. [31] who compared intraperitoneal moxifloxacin and vancomycin treatment for periprosthetic infection in rats. Similarly, they found equal moxifloxacin bone concentrations between infected and healthy bone, as opposed to vancomycin. However, the present authors were not able to document a potential cure of the infected tibias due to lack of cultures in Group C animals. This could seriously question the conclusion for the absence of any disparity between healthy and infected bone samples.
The composite histology score was significantly different between Groups A and B on days 7 and 21 of sacrifice. Grading was clearly higher for Group A for the first 5 weeks. The greater differences observed during the first 3 weeks could be attributable, in part, to the higher acute infection grades of control animals compared with animals under treatment. Interestingly, differences were smoothed over the next 3 weeks as signs of acute infection generally subsided. This may be explained by the potentially longer time period required for cellular infiltration to resolve in the setting of chronic bone infections [32].
Systemic drug toxicity was not evaluated in the present study. It is well known that systemically administered newer fluoroquinolones, such as moxifloxacin, have been associated with a variety of low-incidence drug-related adverse reactions despite the fact that they have an enhanced safety profile [28,29]. Taking into account the long treatment period
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required for osteomyelitis, potential drug toxicity could question clinical safety and should not be ignored. However, the low dose used in this study made the chance of adverse effects rather unlikely.
5. Conclusion These findings provide further and sophisticated treatment modalities against chronic bone infections. IM moxifloxacin treatment was found to be effective in an experimental model of MRSA osteomyelitis. This therapeutic efficacy, along with the aforementioned favourable PK properties of moxifloxacin, render this protocol a potent treatment strategy for osteomyelitis. However, future studies should be conducted to confirm and validate these results on susceptible bacteria in a clinical setting.
Acknowledgements The authors thank the staff of the research laboratory of the 4th Department of Internal Medicine of Athens Medical School and the Experimental & Research Centre of ELPEN Pharmaceutical Co. Inc. for their help and dedication to this project.
Funding: This study was supported by ELPEN Pharmaceutical Co. Inc., Athens, Greece; and Bayer Hellas AG, Athens, Greece.
Competing interests: EJGB has received independent educational grants from Abbott (Hellas) SA and from Wyeth/Ayest SA. The other authors have nothing to disclose.
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Ethical approval: This study was approved by the Veterinary Directorate of the Prefecture of Athens according to Greek legislation in conformance with Directive 2010/63 of the European Union, and followed ethics of experimental animal model studies.
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[24] San Juan R, Garcia-Reyne A, Caba P, Chaves F, Resines C, Llanos F, et al. Safety and efficacy of moxifloxacin monotherapy for treatment of orthopedic implant-related staphylococcal infections. Antimicrob Agents Chemother 2010;54:5161–6. [25] Ang JY, Asmar BI. Multidrug-resistant viridans streptococcus (MDRVS) osteomyelitis of the mandible successfully treated with moxifloxacin. South Med J 2008;101:539–40. [26] Frippiat F, Meunier F, Derue G. Place of newer quinolones and rifampicin in the treatment of Gram-positive bone and joint infections. J Antimicrob Chemother 2004;54:1158; author reply 1159. [27] Metzler K, Hansen GM, Hedlin P, Harding E, Drlica K, Blondeau JM. Comparison of minimal inhibitory and mutant prevention drug concentrations of 4 fluoroquinolones against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus. Int J Antimicrob Agents 2004;24:161–7. [28] Goldstein EJC, Citron DM, Warren YA, Tyrrell KL, Rybak MJ. Virulence characteristics of community-associated Staphylococcus aureus and in vitro activities of moxifloxacin alone and in combination against community-associated and healthcareassociated meticillin-resistant and -susceptible S. aureus. J Med Microbiol 2008;57:452– 6. [29] von Eiff C, Peters G. Comparative in-vitro activities of moxifloxacin, trovafloxacin, quinupristin/dalfopristin and linezolid against staphylococci. J Antimicrob Chemother 1999;43:569–73. [30] Mader JT, Adams K, Morrison L. Comparative evaluation of cefazolin and clindamycin in the treatment of experimental Staphylococcus aureus osteomyelitis in rabbits. Antimicrob Agents Chemother 1989;33:1760–4.
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[31] Beckmann J, Kees F, Schaumburger J, Kalteis T, Lehn N, Grifka J, et al. Tissue concentrations of vancomycin and moxifloxacin in periprosthetic infection in rats. Acta Orthop 2007;78:766–73. [32] Gillespie WJ, Allardyce RA. Mechanisms of bone degradation in infection: a review of current hypotheses. Orthopedics 1990;13:407–10. 10
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Fig. 1. Bacterial growth [mean ± standard error (SE) log10 colony-forming units/g] of test pathogen on animal sacrifice. Group A, no treatment (control); Group B, intramuscular treatment. P<0.05 on days 14, 21, 28 and 42 between Groups A and B.
a
Number shown in
parentheses is the true day of sacrifice for Group B animals, taking into account the 2-week interval when they did not receive antibiotics.
11
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Fig. 2. Composite histology score [mean ± standard error (SE)] per day of sacrifice. Group A, no treatment (control); Group B, intramuscular treatment. P<0.05 on days 7 and 21 between Groups A and B.
a
Number shown in parentheses is the true day of sacrifice for Group B
animals, taking into account the 2-week interval when they did not receive antibiotics.
12
22 Page 22 of 23
13 14
Table 1. Local median (interquartile range) moxifloxacin concentrations (μg/g) in cortical bone samples of Group C animals. Sampling site Infected tibial bone Healthy tibial bone
Day of sacrifice 7
14
35
42
0.66 (0.02)
0.64 (0.035)
0.65 (0.005)
0.64 (0)
0.65 (0.005)
0.65 (0.005)
0.65 (0.015)
0.65 (0.005)
15 16 17 18 19
23 Page 23 of 23
S0924-8579(17)30175-9 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.041 ANTAGE 5121
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
4-10-2016 28-1-2017
Please cite this article as: Vasileios Soranoglou, Ilias Galanopoulos, Evangelos J. GiamarellosBourboulis, Apostolos Papalois, Efthymia Giannitsioti, Lazaros A. Poultsides, Theodosia Choreftaki, Kyriaki Kanellakopoulou, Efficacy of intramuscular moxifloxacin in the treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Efficacy of intramuscular moxifloxacin in the treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus
Vasileios Soranoglou a,*, Ilias Galanopoulos b, Evangelos J. Giamarellos-Bourboulis c, Apostolos Papalois d, Efthymia Giannitsioti c, Lazaros A. Poultsides e, Theodosia Choreftaki f, Kyriaki Kanellakopoulou c
a
2nd Department of Orthopaedics, Athens General Hospital, Athens, Greece
b
1st Department of Orthopaedics, General Military Hospital, Athens, Greece
c
4th Department of Internal Medicine, University of Athens, Medical School, Athens, Greece
d
ELPEN Pharmaceutical Company, Pikermi, Greece
e
Adult Reconstruction and Joint Replacement Division, Department of Orthopaedic Surgery,
Hospital for Special Surgery, New York, USA f
Department of Pathology, Athens General Hospital, Athens, Greece
*
Corresponding author. Present address: Adult Reconstruction and Joint Replacement
Division, Department of Orthopaedic Surgery, Hospital for Special Surgery, 535 East 70th St, 10021, New York, USA. E-mail address: [email protected] (V. Soranoglou).
ARTICLE INFO Received 4 October 2016 Accepted 28 January 2017
Keywords: 1 Page 1 of 23
Moxifloxacin Osteomyelitis treatment Intramuscular Bone concentration MRSA
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1
Highlights
2
osteomyelitis model.
3 4
Intramuscular moxifloxacin treatment proved effective in an experimental
Moxifloxacin bone levels were approximately 43 times higher than the minimum
5
inhibitory
6
Staphylococcus aureus.
7 8
concentration
for
the
tested
strain
of
methicillin-resistant
No differences in moxifloxacin bone levels were found between healthy and infected tibias.
9 ABSTRACT Fluoroquinolones have been well studied in the treatment of chronic osteomyelitis due to their beneficial pharmacokinetic (PK) and pharmacodynamic profiles. The purpose of this study was to determine the efficacy of intramuscular (IM) moxifloxacin administration in the treatment of experimental osteomyelitis by methicillin-resistant Staphylococcus aureus.
Following an experimental osteomyelitis animal model described previously, three groups of rabbits (A=control; B=IM moxifloxacin administration; C=PK study of moxifloxacin penetration into bone) were evaluated. Three weeks after bacterial inoculation, surgical debridement was performed in all animals and IM treatment commenced for Groups B and C. Sacrifice was performed in an A:B group animal ratio of 1:2 at weekly intervals from 7th to 42nd day post debridement and from 21st to 56th day post debridement for Groups A and B, respectively (including 2-week interval without antibiotics for Group B). Cancellous bone was harvested for microbiological and histopathological analyses at re-operation and sacrifice for Groups A and B. Cortical bone moxifloxacin levels were measured in Group C following 7, 14, 35 and 42 days of treatment.
In Group A, bacterial growth after surgical debridement was significant, whereas high eradication rates were observed in Group B. Radiological abnormalities and histopathological 3 Page 3 of 23
findings were evaluated. Moxifloxacin bone levels, observed in Group C, were approximately 43 times greater than the minimum inhibitory concentration, with no difference found between infected and healthy tibial bone.
The therapeutic protocol was very effective in this model of experimental osteomyelitis. However, further evaluation of these results in clinical studies is crucial. 1. Introduction Chronic osteomyelitis is one of the most difficult infections to treat in humans [1]. Treatment requires accurate identification of the causative micro-organism and sensitivity to the antimicrobial agents. Classical documented therapeutic options include intravenous (IV) administration of antimicrobial agents for at least 4–6 weeks along with operative intervention. The latter may include radical surgical debridement, elimination of dead space, bone and soft tissue reconstruction, and skin coverage with grafts [1–6]. However, despite the well-established treatment protocols, significant recurrence rates are still reported in the literature [7]. Inability to achieve high local antimicrobial concentrations in the infected bone and surrounding soft tissue area following systemic administration is one of the factors that may compromise the definitive treatment of chronic osteomyelitis.
Fluoroquinolones, particularly ciprofloxacin, are used in the treatment of osteomyelitis due to their excellent activity against most common pathogens and low systemic toxicity. Furthermore, they show a favourable pharmacokinetic (PK) profile, consisting of high bioavailability (60–99%), moderate protein binding (20–60%) and good tissue penetration [8]. Additionally, their ability to penetrate into cells may be advantageous in bone infections, as in-vitro studies have shown that Staphylococcus aureus can penetrate into and survive in osteoblasts [9].
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Moxifloxacin, a fourth-generation fluoroquinolone, appears to be a promising, clinically safe agent with beneficial features in the era of bone infections [9–13]. Its long elimination halflife of 13 h means that it can be dosed daily, whereas other antimicrobial agents (e.g. ciprofloxacin, vancomycin, clindamycin) require more frequent administration. A systematic review of antibiotic penetration into bone [9] showed higher bone:serum concentration ratios for moxifloxacin (mean range 0.33–1.05) compared with glycopeptides (mean range 0.1–0.6), clindamycin (mean range 0.21–0.45) and rifampicin (mean range 0.08–0.57). Additionally, moxifloxacin demonstrates excellent bioavailability after oral administration [13], comparable with that of clindamycin [14]. This is extremely valuable when considering the long duration of systemic therapeutic regimens for osteomyelitis that usually require central venous catheters for parenteral administration. Appropriately chosen oral agents demonstrate clinical equivalence compared with parenteral osteomyelitis therapy [15].
This study evaluated an intramuscular (IM) moxifloxacin therapeutic protocol in the setting of an established experimental model of methicillin-resistant S. aureus (MRSA) osteomyelitis. The aim of this study was to assess: (i) cure or eradication rates of the infection by parenteral treatment; and (ii) evaluation of bone moxifloxacin levels in systemically treated subjects.
2. Materials and methods 2.1. Study design One MRSA experimental osteomyelitis model, modified from that described previously by Norden and Andriole [16–20], was utilized. Three study groups were formed: A (control, n=18), B (IM moxifloxacin therapy, n=36) and C (PK study of IM moxifloxacin bone
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penetration, n=12). To avoid sacrifice of an extreme number of animals, it was considered that PK studies including animals at a 1:3 ratio of the treatment group would be sufficient.
2.2. Animal model In total, 66 white New Zealand male rabbits (body weight 2.3–3.6 kg) were included in the study. Animals were housed in single metal cages and had access to tap water and standard balanced rabbit chow ad libitum. Room temperature ranged between 18 ◦C and 22 ◦C, relative humidity ranged between 55% and 65%, and the light/dark cycle was 06:00/18:00 h.
2.3. Pathogen The MRSA isolate was derived from a patient who presented with chronic osteomyelitis. The minimum inhibitory concentration (MIC) of moxifloxacin was determined by a microdilution technique in 96-well plates using a 5x105 colony-forming units (cfu)/mL log-phase inoculum at a final volume of 0.1 mL in Mueller–Hinton broth (Becton-Dickinson, Franklin Lakes, NJ, USA); this was ≤0.015 μg/mL. After growth for 5 h at 37 ◦C in a shaking water bath in Mueller–Hinton broth, a 1×108 cfu/mL inoculum was prepared and applied for bacterial challenge, as described below.
2.4. Antibacterial agent Pure moxifloxacin hydrochloride powder (Bayer Healthcare AG, Berlin, Germany) was admixed with sterile water for injections BP (B. Braun Melsungen AG, Melsungen, Germany) and vortex stirred to a sterile 2% aqueous solution (20 mg/mL) which was used for the IM injections. The solution was prepared daily. Unused solution and solutions with visible undissolved particles were discarded. The selected dose was 5 mg/kg body weight once per day based on the study of Fernández-Varón et al. [21].
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2.5. Experimental animal model assay Osteomyelitis was induced surgically in the right upper tibial metaphysis in all rabbits. Animals were sedated by IM injection of 25 mg/kg body weight ketamine 10% (Νarketan 10, Vetoquinol AG, Belp-Bern, Switzerland) and 5 mg/kg body weight xylazine 2% (Rompun Bayer AG, Leverkusen, Germany). Anaesthesia was maintained by IM administration of 15 mg/kg body weight xylazine at 30-min intervals. A 2-cm skin incision was made 2.5 cm distal to the knee joint, exposing the anteromedial cortex of the proximal tibial metaphysis. Using a 2-mm drill, a hole was made and 0.1 mL of the prepared bacterial suspension was instilled. A 25G needle was placed through the hole, serving as a foreign body. The needle was curved at one end in order to be fixed securely on to the tibial cortex. Finally, the hole was plugged with sterile bone wax.
The infection was allowed to develop for 3 weeks, at which time all animals underwent a second operation for removal of the needle and debridement of necrotic and infected tissue. Cancellous bone samples were also obtained from the site of infection for quantitative cultures, and the hole was plugged with sterile bone wax. Animals were then assigned at random into three groups: Group A (n=18), without any subsequent antimicrobial treatment (control group); Group B (n=36), with initiation of IM moxifloxacin therapy of 5 mg/kg body weight dose q.d.; and Group C (n=12), which received the same treatment as Group B and served as the PK study group. Postoperative analgesia was achieved by IM paracetamol (Apotel, Uni-Ρharma, Pharmaceutical Laboratories S.A., Kifissia, Greece) administration according to standard practice for experimental model assays.
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During the experimental study, animal weight and temperature were assessed weekly and surgical trauma was inspected daily. Three animals in Group A were sacrificed at regular time intervals (i.e. 7, 14, 21, 28, 35 and 42 days after the day of reoperation) by intracardiac pentobarbital sodium 20% (Dolethal, Vetoquinol SA, Lure Cedex, France) injection after sedation. Animals in Group B were treated for periods of 7, 14, 21, 28, 35 and 42 days (six animals in each treatment period), and were sacrificed following a 2-week interval without antibiotics. The rationale behind this interval was to evaluate any potential regrowth of S. aureus bone infection [6]. On sacrifice, infected tibias were harvested and specimens of cancellous bone were obtained under sterile conditions for quantitative culture and biopsy. Animals in Group C were divided into four equal subgroups and received IM treatment for 7, 14, 35 and 42 days, respectively, before sacrifice. Cortical bone samples were obtained from the site of infection and the corresponding site of the healthy tibia 24 h after the last IM dose, and used for measurement of moxifloxacin bone levels.
2.6. Bone cultures Cancellous bone samples harvested during re-operation and upon sacrifice were weighed and homogenized by a sterile tissue grinder. Samples were suspended in 1 mL of sterile saline solution and cultured quantitatively by seven consecutive 1:10 dilutions in sterile water. A 0.1-mL aliquot of each dilution was then plated on to McConkey agar (Becton Dickinson, Cockeysville, MD, USA). This serial subculturing allowed avoidance of any antibiotic carryover effect. Bacterial growth was expressed as cfu/g tissue. The lowest detection limit was 10 cfu/g.
2.7. Histopathology Separate bone particles harvested on sacrifice from Group A and B animals were fixed in neutral buffered formalin, embedded in paraffin wax, sectioned and stained with 8 Page 8 of 23
haematoxylin and eosin. A semiquantitative scoring system, adopted with modifications from previous studies [20,22], was used to grade acute and chronic inflammation, and giant cell infiltration. The first two parameters were scored as 0 (absent), 1 (sparse), 2 (moderate) and 3 (intense). Giant cell infiltration was graded as 1 (positive) or 0 (negative). As all histological parameters were given equal diagnostic weight, a composite (arithmetic sum) score was calculated for each animal and used for the comparative evaluation.
2.8. Radiology Plain anteroposterior and lateral radiographs of the infected tibia were taken twice for each animal in Groups A and B using exposure factors of 36 keV and 25 ms. The first radiograph was taken just before re-operation and the second radiograph was taken at the completion of treatment just before animal sacrifice. During x-rays, animals were sedated and small sandbags were used to immobilize the leg in the proper position.
2.9. PK study of moxifloxacin bone penetration The grinding procedure was repeated for each sample of cortical bone collected from the two corresponding sites in Group C. Adopting a technique described previously [20], samples were centrifuged for 10 min at 1400×g at 4 ◦C, after suspension of 1 mL of sterile sodium saline. Supernatants were collected and stored at −80 ◦C. Moxifloxacin concentrations were estimated in bone supernatants by high-performance liquid chromatography (HPLC). Briefly, 250 μL of sample was diluted with 250 μL of displacing reagent and centrifuged for 5 min at 2700×g. The displacing reagent consisted of 0.5% sodium dodecyl sulphate (SDS) (Merck, Darmstadt, Germany) and 20% acetonitrile (Merck) in 75mM sodium phosphate buffer (pH 7.5). Ten microlitres of the supernatant were injected into an HPLC system (Agilent 1100 Series; Agilent Technologies, Waldbronn, Germany) with the following elution
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characteristics: Nucleosil 100-5 C18 (4.6 mm×250 mm, 5 μm) column at 37 ◦C; mobile phase consisting of a pH 3.0 buffer composed of 25 mM Na3PO4 and 10 mM SDS and acetonitrile 99% at a 35/65 ratio with a flow rate of 1 mL/min; and ultraviolet detection at 293 nm. The retention time of moxifloxacin was 2.9 min and its concentration was estimated (in μg/mL) using a standard curve ranging between 0.095 and 100 μg/mL. All determinations were performed in duplicate. R2 of the curve was 0.9997. The interday variation of the assay was 4% [20].
2.10. Statistical analysis Bacterial growth and histological scores were expressed as mean ± standard error, and drug levels were expressed as median and interquartile range. Comparisons between groups were performed using analysis of variance with post-hoc Bonferroni corrections. Statistical significance was set at P<0.05.
3. Results The mean log10 ± standard deviation of bacterial growth in cancellous bone of all tested animals upon re-operation before assignment into each of the three groups (A, B, and C) was 4.44±1.38 cfu/g. Bacterial growth from cancellous bone of Group B animals was below the detection limit after 28 and 42 days of treatment, and was lower than the respective growth in Group A at all days of sacrifice. This difference between Group A and Group B upon sacrifice was significant at 14 days (P=0.015), 21 days (P=0.011), 28 days (P=0.006) and 42 days (P=0.006) (Fig. 1). After ≥28 days of treatment, bacterial regrowth was only documented in one of 18 sacrificed animals in Group B. Eradication rates were ≥67% on all sacrifice days in Group B, whereas negative culture results upon sacrifice were only documented in one animal in Group A.
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Clinical examination of infected animals before their second operation revealed various degrees of localized or diffuse limb inflammation. Increased local temperature, abscesses and pus draining fistulas were the main macroscopic findings. The clinical image was significantly improved, as expected, after osteomyelitis treatment in Group B. Radiographic signs of osteomyelitis were observed 3 weeks after infection. These included periosteal elevation, local demineralization and cortical irregularity with radiolucent areas. At sacrifice, radiographs in Group A revealed intense radiologic abnormalities and formation of sequestra in four animals. However, in Group B, the radiologic abnormalities were less obvious, with cortical thickening and sclerosis being the leading signs.
Comparative evaluation of the histopathological composite scores between Groups A and B revealed lower composite scores for Group B for the first 5 weeks of treatment (Fig. 2). However, differences only reached statistical significance on days 7 and 21 of sacrifice. In Group A, the grading of acute infection outweighed chronic infection for the first 3 weeks, whereas chronic infection was prevalent in Group B for the same period. Bone concentrations of moxifloxacin in cortical bone samples from infected and healthy tibias of Group C are shown in Table 1. Interestingly, almost identical drug levels were measured in healthy and infected bone samples.
4. Discussion
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The use of fluoroquinolones in the treatment of chronic osteomyelitis has been well documented over the last two decades. Parenteral fluoroquinolone administration along with surgical debridement and bone reconstruction remains an excellent treatment protocol for management. Among the fluoroquinolones, moxifloxacin shows excellent bioavailability, a long half-life and superior bone penetration [9]. Multiple in vitro and in vivo studies have shown the efficacy of moxifloxacin against Gram-positive pathogens. These studies demonstrated superiority against Staphylococcus spp. compared with older-generation fluoroquinolones, such as ciprofloxacin [23]. Moreover, the risk of resistance development during monotherapy is theoretically lower for moxifloxacin [24]. However, in the clinical setting, there are few reports for treatment of bacterial osteomyelitis and periprosthetic infection with this antibiotic [24–26].
To the best of the authors’ knowledge, no published data exist regarding the efficacy of this antimicrobial agent in IM treatment of osteomyelitis. The rationale behind using the IM route rather than the IV route was based on the fact that IV drug administration in rabbits is extremely challenging due to the lack of available veins, and it is not possible to maintain an intravenous catheter for a long time in a vigilant rabbit. Moreover, IM administration of antibiotics can result in peak serum concentrations within minutes at levels comparable to those observed after IV injections [22,23].
The study results showed that this therapeutic protocol was highly effective. After ≥4 weeks of treatment, only one of 18 animals had persistent infection. Eradication rates were ≥67% after just 1 week of treatment. A noteworthy aspect of this study was the 2-week interval between end of treatment and sacrifice. This strategy, uncommon in the literature, provided the opportunity to evaluate potential recurrence of infection. These findings should guide
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future clinical studies for further evaluation of systemic moxifloxacin administration in osteomyelitis. Furthermore, the simple dosing protocol (once per day) and safety profile of this antibiotic could overcome patient compliance issues.
A low dose (5 mg/kg body weight) of moxifloxacin was used in this study because of the very low MIC of the MRSA strain. A higher dose would possibly achieve even higher eradication rates in earlier time points. Fernández-Varón et al. [21] described moxifloxacin PK after IV, IM and oral administration in rabbits and concluded that a 5 mg/kg body weight dose would be effective against bacterial isolates with MIC ≤0.06 μg/mL. For an MIC of 0.06 μg/mL, the 24-h area under the concentration-time curve/MIC ratio was 97 h (ΙΜ). The maximum serum concentration (Cmax)/MIC ratio was approximately 10. Typically, a Cmax/MIC ratio of 8–10 has been associated with effective treatment of Gram-negative and -positive bacterial infections and prevention of resistance [27]. In this study, the MIC was ≤0.015 μg/mL.
This study also found that trough bone concentrations of moxifloxacin following IM administration were approximately 43 times higher than the MIC of moxifloxacin for the specific pathogen. However, the bacterial isolate used in this study had a very low MIC compared with strains in clinical practice, where MIC90 values (MIC for 90% of the organisms) of moxifloxacin can be up to 8 μg/mL, especially for hospital-acquired infections [27–29]. A much higher IM dose would be necessary in order to achieve therapeutic bone concentrations, but the required solution volume would be inappropriate for IM administration in rabbits.
Another important therapeutic consideration is the effect of inflammation on the penetration of antibiotic into bone. In osteomyelitis, blood flow into bone may be increased because of
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reactive hyperaemia in the infected area, or decreased due to the presence of pus and sequestra. Most of the bone penetration studies have been conducted in patients with uninfected bone [9]. In a study of osteomyelitis in rabbits, Mader et al. [30] found bone/serum concentration ratios of 0.98 for infected bone and 0.40 for uninfected bone after an IV dose of clindamycin 70 mg/kg body weight. No differences were documented in the present PK study. This is in accordance with the results of Beckmann et al. [31] who compared intraperitoneal moxifloxacin and vancomycin treatment for periprosthetic infection in rats. Similarly, they found equal moxifloxacin bone concentrations between infected and healthy bone, as opposed to vancomycin. However, the present authors were not able to document a potential cure of the infected tibias due to lack of cultures in Group C animals. This could seriously question the conclusion for the absence of any disparity between healthy and infected bone samples.
The composite histology score was significantly different between Groups A and B on days 7 and 21 of sacrifice. Grading was clearly higher for Group A for the first 5 weeks. The greater differences observed during the first 3 weeks could be attributable, in part, to the higher acute infection grades of control animals compared with animals under treatment. Interestingly, differences were smoothed over the next 3 weeks as signs of acute infection generally subsided. This may be explained by the potentially longer time period required for cellular infiltration to resolve in the setting of chronic bone infections [32].
Systemic drug toxicity was not evaluated in the present study. It is well known that systemically administered newer fluoroquinolones, such as moxifloxacin, have been associated with a variety of low-incidence drug-related adverse reactions despite the fact that they have an enhanced safety profile [28,29]. Taking into account the long treatment period
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required for osteomyelitis, potential drug toxicity could question clinical safety and should not be ignored. However, the low dose used in this study made the chance of adverse effects rather unlikely.
5. Conclusion These findings provide further and sophisticated treatment modalities against chronic bone infections. IM moxifloxacin treatment was found to be effective in an experimental model of MRSA osteomyelitis. This therapeutic efficacy, along with the aforementioned favourable PK properties of moxifloxacin, render this protocol a potent treatment strategy for osteomyelitis. However, future studies should be conducted to confirm and validate these results on susceptible bacteria in a clinical setting.
Acknowledgements The authors thank the staff of the research laboratory of the 4th Department of Internal Medicine of Athens Medical School and the Experimental & Research Centre of ELPEN Pharmaceutical Co. Inc. for their help and dedication to this project.
Funding: This study was supported by ELPEN Pharmaceutical Co. Inc., Athens, Greece; and Bayer Hellas AG, Athens, Greece.
Competing interests: EJGB has received independent educational grants from Abbott (Hellas) SA and from Wyeth/Ayest SA. The other authors have nothing to disclose.
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Ethical approval: This study was approved by the Veterinary Directorate of the Prefecture of Athens according to Greek legislation in conformance with Directive 2010/63 of the European Union, and followed ethics of experimental animal model studies.
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[24] San Juan R, Garcia-Reyne A, Caba P, Chaves F, Resines C, Llanos F, et al. Safety and efficacy of moxifloxacin monotherapy for treatment of orthopedic implant-related staphylococcal infections. Antimicrob Agents Chemother 2010;54:5161–6. [25] Ang JY, Asmar BI. Multidrug-resistant viridans streptococcus (MDRVS) osteomyelitis of the mandible successfully treated with moxifloxacin. South Med J 2008;101:539–40. [26] Frippiat F, Meunier F, Derue G. Place of newer quinolones and rifampicin in the treatment of Gram-positive bone and joint infections. J Antimicrob Chemother 2004;54:1158; author reply 1159. [27] Metzler K, Hansen GM, Hedlin P, Harding E, Drlica K, Blondeau JM. Comparison of minimal inhibitory and mutant prevention drug concentrations of 4 fluoroquinolones against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus. Int J Antimicrob Agents 2004;24:161–7. [28] Goldstein EJC, Citron DM, Warren YA, Tyrrell KL, Rybak MJ. Virulence characteristics of community-associated Staphylococcus aureus and in vitro activities of moxifloxacin alone and in combination against community-associated and healthcareassociated meticillin-resistant and -susceptible S. aureus. J Med Microbiol 2008;57:452– 6. [29] von Eiff C, Peters G. Comparative in-vitro activities of moxifloxacin, trovafloxacin, quinupristin/dalfopristin and linezolid against staphylococci. J Antimicrob Chemother 1999;43:569–73. [30] Mader JT, Adams K, Morrison L. Comparative evaluation of cefazolin and clindamycin in the treatment of experimental Staphylococcus aureus osteomyelitis in rabbits. Antimicrob Agents Chemother 1989;33:1760–4.
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[31] Beckmann J, Kees F, Schaumburger J, Kalteis T, Lehn N, Grifka J, et al. Tissue concentrations of vancomycin and moxifloxacin in periprosthetic infection in rats. Acta Orthop 2007;78:766–73. [32] Gillespie WJ, Allardyce RA. Mechanisms of bone degradation in infection: a review of current hypotheses. Orthopedics 1990;13:407–10. 10
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Fig. 1. Bacterial growth [mean ± standard error (SE) log10 colony-forming units/g] of test pathogen on animal sacrifice. Group A, no treatment (control); Group B, intramuscular treatment. P<0.05 on days 14, 21, 28 and 42 between Groups A and B.
a
Number shown in
parentheses is the true day of sacrifice for Group B animals, taking into account the 2-week interval when they did not receive antibiotics.
11
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Fig. 2. Composite histology score [mean ± standard error (SE)] per day of sacrifice. Group A, no treatment (control); Group B, intramuscular treatment. P<0.05 on days 7 and 21 between Groups A and B.
a
Number shown in parentheses is the true day of sacrifice for Group B
animals, taking into account the 2-week interval when they did not receive antibiotics.
12
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13 14
Table 1. Local median (interquartile range) moxifloxacin concentrations (μg/g) in cortical bone samples of Group C animals. Sampling site Infected tibial bone Healthy tibial bone
Day of sacrifice 7
14
35
42
0.66 (0.02)
0.64 (0.035)
0.65 (0.005)
0.64 (0)
0.65 (0.005)
0.65 (0.005)
0.65 (0.015)
0.65 (0.005)
15 16 17 18 19
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