Treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus with a biodegradable system of lactic acid polymer releasing pefloxacin

Kyriaki Kanellakopouloua, Nearchos Galanakisb, Evangelos J. Giamarellos-Bourboulisc, Christos Rifiotisd, Konstantinos Papakostasd, Andreas Andreopoulose, Eleftherios Dounisd, Panagiotis Karagianakosf and Helen Giamarelloua,*

a 4th Department of Internal Medicine, Athens Medical School; b 2nd Department of Internal Medicine, Nikaia General Hospital; c 1st Department of Propaedeutic Medicine, Athens Medical School; d Department of Orthopaedics, Athens Laiko General Hospital; e Department of Chemical Engineering, National Polytechnic School, Athens; f Department of Experimental Surgery, Athens Medical School, Athens, Greece


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
A novel biodegradable system of D-,L-dilactide delivering pefloxacin was implanted in 104 rabbits with experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus (MRSA), 26 serving as controls. Animals were killed on each third day and viable bacterial counts and levels of pefloxacin in bone tissue were determined. A 99.9% decrease in viable count of bacteria was achieved by day 12 and complete bacterial eradication on day 33. Pefloxacin was released gradually, reaching its peak on day 15 at levels 100 times the MIC of pefloxacin for MRSA. The biodegradable system described may have a future role in the therapeutic approach to osteomyelitis.


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Management of chronic osteomyelitis with the local delivery of the antimicrobial agent is a novel therapeutic modality, which achieves elevated antibiotic concentrations at the site of infection without systemic toxicity.1 Biodegradable systems for local release of fluoroquinolones have been evolved to replace the polymethylmethacrylate (PMMA) cement beads carrying gentamicin or tobramycin in an effort to overcome the need for surgical removal upon completion of antibiotic release.2 A similar system for the release of fluoroquinolones has been described by our study group:3 a polymer of D-,L-dilactide which has been shown to elute elevated concentrations of ciprofloxacin and pefloxacin for 50–350 days in vitro. This polymer has been evaluated for the in vivo release of pefloxacin and the treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus (MRSA).


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Bacterial isolate

A clinical isolate of S. aureus from a patient with chronic osteomyelitis and with an MIC of pefloxacin of 0.5 mg/L was used. Resistance to methicillin was defined after growth on an oxacillin agar plate.4 After overnight growth an inoculum of 3.7 x 107 cfu/mL was prepared using appropriate dilutions of the 2.0 McFarland standard.

Lactic acid polymer

The polymer was produced as described previously3 by the direct polymerization of lactic acid using Sn(Oct)2 as a catalyst. Pefloxacin (Rhône Polulenc Rorer, Vitry-sur-Seine, France) was mixed at a ratio of 1:10.

Animal groups

A total of 104 male rabbits weighing 2.9–3.5 kg each was used (26 controls and 78 treated by implantation of the polymer).

Osteomyelitis model

Osteomyelitis was produced in the left tibia of the rabbits by the modified Norden model5. After anaesthesia with nembutal 0.5 mg/kg iv, a 2 cm incision was made 2.5 cm below the condyles of the inner tibia to expose its upper third. A 2 mm hole was drilled in the bone medulla where a suspension of 0.1 mL containing the bacterial inoculum was instilled. A thin needle of 25G serving as a foreign body was placed through the hole which was closed with a sterile bone wax. Postoperative analgesia was achieved by the administration of suppositories of paracetamol.

Follow-up of the infection was performed by measuring rectal temperature and body weight and by tibial X-rays as in clinical practice. On the third week all animals were reoperated on and the needle was removed, followed by implantation of the polymer.

Microbiological evaluation and pharmacokinetic study

Two contol and six treated animals were killed at 24 h, on each third day and on day 47. Samples were selected from blood, cortical bone at the site of implantation and juxtaposed to it and at 1 and 2 cm from the polymer, the opposite tibia, the spongiosa bone and the neighbouring skin and muscle. All remnants of the implanted polymer were also removed for measurement of the remaining pefloxacin. Tissue samples were washed thoroughly to remove blood, weighed and homogenized. Homogenized tissue was suspended in 2 mL of sterile saline solution and quantitively cultured by seven consecutive 1:10 dilutions in sterile water and plating on to blood agar (Becton Dickinson, Cockeysville, MD, USA). Bacterial growth was expressed as colony-forming units (cfu)/g bone tissue. The limit of viable cell count detection was 10 cfu/g bone. Changes in viable count were expressed as their mean log10 increase or decrease on each day of sampling compared with the implanted inoculum. Levels of pefloxacin were determined by a microbiological agar well diffusion assay using Mueller– Hinton agar (Becton Dickinson) and Escherichia coli strain ICB 4004 as the indicator microorganism after plotting a standard curve of known concentrations of pefloxacin. Mean values (± s.d.) were determined on each day of sampling.


    Results
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 Material and methods
 Results
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Clinical evidence of infection including fever and loss of body weight over the first 3 weeks post-removal of the needle and radiological signs of periosteal reaction, cortical erosion and new bone formation were documented in all animals. Two animals belonging to the control group died before removal of the thin needle. Changes in viable cell count in treated and control animals are presented in the FigureGo. The pharmacokinetics of pefloxacin release are shown in the TableGo. Pefloxacin remained undetected in the surrounding tissues, in the opposite tibia, in spongiosa bone and in the blood of the animals studied for the whole of the sampling period.



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Figure. Changes in the inoculum of methicillin-resistant Staphylococcus aureus after implantation of a lactic acid polymer releasing pefloxacin: ({blacksquare}), treated animals; ({square}), controls.

 

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Table. Pharmacokinetics of the release of pefloxacin from the implanted lactic acid polymer (mean ± s.d., µg/g of tissue) at different sites near the area of implantation
 

    Discussion
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 Material and methods
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The novel biodegradable carrier of pefloxacin proved very effective in the eradication of bacteria as demonstrated by the changes in the cell counts of viable MRSA in both study and control groups (FigureGo). Bacterial killing, usually expressed as a 99.9% decrease of the implanted inoculum,4 was achieved by day 12, reaching a 6 log10 decrease synonymous with bacterial sterility by day 33. Minimal changes in the implanted inoculum were seen in the control group over these periods approaching increases of 0.5 log10.

Similar results in the treatment of canine staphylococcal osteomyelitis were reported by Garvin et al.6 with a polylactide/polyglycolide delivery system of gentamicin as well as by Wei et al.7 with an oligomer of lactic acid carrying dideoxykanamycin B in rabbits, but these studies involved isolates susceptible to methicillin. Successful clinical data on osteomyelitis of the pelvis and of the hip caused by MRSA exist in only two patients after the application of PMMA beads impregnated with vancomycin.8 As a consequence the application of the biodegradable polymer described for the therapy of osteomyelitis by MRSA might be a novel therapeutic modality.

The reason for the efficacy of the lactic acid polymer in the treatment of MRSA osteomyelitis is probably the advantageous pharmacokinetics at the site of infection (TableGo). The polymer achieved a gradual release of pefloxacin and was completely disintegrated by day 33. However, no bacterial regrowth was documented after the disintegration of the polymer as seen by the persistence of bone sterility on day 47 (FigureGo). The gradual release of pefloxacin was accompanied by a gradual increase in its levels just near and around the site of implantation of the polymer, reaching its peak on day 15 and then steadily decreasing. That peak approaches concentrations 100 times the MIC of pefloxacin for the isolate of MRSA used. It should be emphasized that diffusion of pefloxacin from the 2 kDa polymer is advantageous compared with work published using the PMMA beads, which permitted the in vivo elution of ciprofloxacin for 28 days and of tobramycin for only 1 week.9

The present study revealed that the local treatment of experimental osteomyelitis caused by MRSA with a low molecular weight polymer containing pefloxacin results in the eradication of MRSA. Its effectiveness, accompanied by the lack of systemic toxicity, and the property of biodegradation, which avoids the necessity for surgical removal upon completion of antibiotic release, strongly support the future evolution of such delivery systems of quinolones for the treatment of chronic osteomyelitis.


    Acknowledgments
 
Presented in part at the Thirty-Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, 1995 San Francisco, CA (abstract no. B1). American Society for Microbiology, Washington, DC.


    Notes
 
* Correspondence address. 4th Department of Internal Medicine, Sismanoglio General Hospital, 15126 Maroussi Attikis, Greece. Tel: +301-80-39-542/+301-80-33-817; Fax: +301-80-39-543. Back


    References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
1 . Walenkamp, G. H. (1997). Chronic osteomyelitis. Acta Orthopedica Scandinavica 68, 497–506.

2 . Nelson, C. L., Griffin, F. M., Harrison, B. H. & Cooper, R. E. (1992). In vitro elution characteristics of commercially and noncomercially prepared antibiotic PMMA beads. Clinical Orthopaedics and Related Research 284, 303–9.[Medline]

3 . Kanellakopoulou, K., Kolia, M., Anastassiadis, A., Korakis, T., Giamarellos-Bourboulis, E. J., Andreopoulos, A. et al. (1999). Lactic acid polymers as biodegradable carriers of fluoroquinolones: an in vitro study. Antimicrobial Agents and Chemotherapy 43, 714–16.[Abstract/Free Full Text]

4 . Woods, G. L. & Washington, J. A. (1995). Antibacterial susceptibility tests: dilution and disk diffusion methods. In Manual of Clinical Microbiology, 6th edn, (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 1327–41. American Society for Microbiology, Washington, DC.

5 . Norden, C. W. (1970). Experimental osteomyelitis. I. A description of the model. Journal of Infectious Diseases 122, 410–18.[ISI][Medline]

6 . Garvin, K. L., Miyano, J. A., Robinson, D., Giber, D., Novak, J., Radio, S. et al. (1994). Polylactide/polyglycolide antibiotic implants in the treatment of osteomyelitis. A canine model. Journal of Bone and Joint Surgery 76A, 1500–6.[Medline]

7 . Wei, C., Kotoura, Y., Oka, M., Yamamuro, T.,Wada, R., Hyon, S. H. et al. (1991). A bioabsorbable delivery system for antibiotic treatment of osteomyelitis: the use of lactic acid oligomer as a carrier. Journal of Bone and Joint Surgery 73B, 246–52.[ISI]

8 . Ozaki, T., Yoshitaka, T., Kunisada, T., Dan'ura, T., Naito, N. & Inoue, H. (1998). Vancomycin-impregnated polymethylmethacrylate beads for methicillin-resistant Staphylococcus aureus (MRSA) infection: report of two cases. Journal of Orthopaedic Science 3, 163–8.[Medline]

9 . Adams, K., Couch, L., Cierny, G., Calhoun, J. & Mader, J. T. (1992). In vitro and in vivo evaluation of antibiotic diffusion from antibiotic-impregnated polymethylmethacrylate beads. Clinical Orthopaedics and Related Research 278, 244–52.[Medline]

Received 9 July 1999; returned 16 December 1999; revised 15 February 2000; accepted 29 February 2000