Efficacy of Fosfomycin in Experimental Osteomyelitis Due to Methicillin-Resistant Staphylococcus aureus

Abstract

The activity of fosfomycin was evaluated in an experimental methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis model. Eighteen rats were treated for 4 weeks with 150 mg of fosfomycin/kg of body weight intraperitoneally once daily or with saline placebo. After treatment, animals were euthanized and the infected tibiae were processed for quantitative bacterial culture. Bone cultures were positive for methicillin-resistant S. aureus in all 9 (100%) untreated controls and in 2 of 9 (22.2%) fosfomycin-treated rats. Thus, fosfomycin treatment was significantly more efficacious than placebo. No development of resistance was observed after the 4-week treatment period.

The rising incidence of infections due to methicillin-resistant Staphylococcus aureus (MRSA) has considerably complicated the treatment of osteomyelitis and raised important issues relating to drug toxicity and development of antibiotic resistance during treatment (5, 26).

Fosfomycin (FOF) is a well-tolerated bactericidal agent with longstanding sensible clinical use (17) and displays broad-spectrum activity against various Gram-positive and Gram-negative bacteria, including difficult-to-treat pathogens such as MRSA (6). It penetrates well into osseous tissue and has proved to be clinically useful in the treatment of acute and chronic osteomyelitis (12, 17, 25). Today, because of concern about development of resistance, FOF is mainly used in combination with other classes of antibiotics (6). The clinical relevance of these findings, however, has been questioned recently (1, 11, 22).

Little is known about the efficacy of FOF monotherapy in chronic osteomyelitis. The aim of the present study was to evaluate the efficacy of such treatment in an experimental rat model of chronic MRSA osteomyelitis.

The study protocol was approved by the local Animal Welfare Committee of the Medical University of Vienna, Vienna, Austria. A clinical isolate of MRSA (4409/07) from a patient with chronic osteomyelitis was used, and the FOF susceptibility of this strain was determined using broth microdilution methods in accordance with the CLSI guidelines.

For in vivo testing, bacteria in exponential growth phase were used. Thus, bacteria grown overnight in Trypticase soy broth (TSB) were diluted 1:100 in TSB and incubated at 37°C for 5 h. A bacterial inoculum containing 1 × 10⁸ to 5 × 10⁸ CFU/ml was prepared, and the number of viable organisms was confirmed from plate counts made before and after the surgical procedure.

Infection of bone was established using a modified version of the model of Zak et al. (27). In total, 30 male Sprague-Dawley rats were used. Animals were anesthetized, and the proximal medial surfaces of the left tibiae were surgically exposed. A 0.1-cm hole was created into the medullary cavity using a high-speed drill. A 20-μl sample of the MRSA inoculum containing 1 × 10⁸ to 5 × 10⁸ CFU/ml was injected into the bone. Drill holes were sealed with sterile bone wax, and the incisions were closed with sutures. Sclerosing agents to facilitate the onset of osteomyelitis were not used in this study.

Three out of 30 rats died during anesthesia and were not further analyzed. Four weeks after infection, 26 of the 27 infected rats (>96%) had radiographically confirmed localized proximal osteomyelitis of the tibia.

For histological confirmation, bone specimens from four animals underwent methylmethacrylate or classic embedding. Semithin sections were colored with Giemsa stain; thin sections were stained with hematoxylin and eosin. The histology of all embedded specimens demonstrated chronic inflammatory changes with osseous destruction and reactive-bone formation, granulation tissue, and bone marrow fibrosis. Moreover, focal intramedullary microabscesses, limited inflammatory cortical ruptures, and small colonies of cocci were found.

Bacterial counts in bone, as described below, were performed in four animals: the counts of S. aureus were 7.58 × 10⁵, 1.30 × 10⁶, 1.81 × 10⁶, and 3.79 × 10⁶ CFU/g of bone.

The 18 remaining rats were randomly assigned to receive either FOF or saline for a further 28 days. FOF powder (Sandoz, Kundl, Austria) was dissolved in sterile water and administered intraperitoneally at a dose of 150 mg/kg of body weight once daily. The rationale for the treatment regimen was to achieve serum levels of drug closely mimicking those obtained in humans (2, 13-15, 19). Twelve hours after completion of antimicrobial therapy, the rats were euthanized. The infected tibiae were aseptically removed, weighed, and pulverized. Sterile physiological saline (10 ml) was added to each specimen, and the bone suspensions were vigorously vortexed. Serial 10-fold dilutions were prepared and incubated on sheep blood agar plates at 37°C for 24 h, and the bacterial colonies were counted. The entire remaining specimen was placed in 10 ml TSB and incubated in 5% CO₂ at 37°C for 48 h. Sterile cultures were assigned a value of 1 log₁₀ CFU/g of bone. FOF MICs for MRSA recovered from bone after FOF treatment were determined according to CLSI guidelines.

The Mann-Whitney U test was used to assess the significance of bacterial clearance and weight variation between the two study groups. Differences between the groups were deemed statistically significant if P < 0.05.

The results of the treatment of experimental MRSA osteomyelitis are shown as log₁₀ CFU/g of bone in Fig. 1. In the placebo group (n = 9), the median bacterial count was 4.67 × 10⁶ CFU/g of bone (range, 4.55 × 10⁴ to 2.24 × 10⁷ CFU/g of bone). In contrast, FOF treatment (n = 9) resulted in sterilized bones in seven out of nine animals; the bacterial counts in the two remaining animals were 7.69 × 10² and 8.47 × 10² CFU/g of bone. The results of treatment with FOF (150 mg/kg body weight) once daily were significantly superior to results in the placebo group (P = 0.0003).

FIG. 1.

Results of fosfomycin (FOF) treatment of experimental MRSA osteomyelitis. Horizontal bars indicate median values for the two groups of rats treated with FOF or placebo. Each symbol represents the value for an individual animal.

The FOF MIC for the clinical strain used in this study was 0.5 μg/ml. The MICs of isolates obtained from the two rats in the FOF treatment group that were still infected at the end of the 4-week treatment period were within 1 dilution of the pretreatment MIC, indicating no emergence of resistance.

The study drug was well tolerated. No adverse effects, such as diarrhea or loss of weight, were observed.

The excellent efficacy of FOF in the treatment of experimental chronic MRSA osteomyelitis may be explained by its favorable osseophilic behavior. Schintler et al. recently demonstrated that the currently approved FOF dosage of 100 mg/kg body weight 2 or 3 times a day results in bone tissue concentrations that work against relevant pathogens, including MRSA (20). This excellent penetration of FOF into osseous tissue becomes particularly important in relation to reports linking the development of bacterial resistance to subinhibitory concentrations of antibiotic at the target site (4, 7, 9).

Nonetheless, because of in vitro studies that revealed the emergence of resistant clones at high frequency, the use of FOF has been largely abandoned in many parts of the world, and today FOF is usually combined with another antibiotic agent to prevent the development of FOF resistance (8, 16). It cannot be emphasized enough that this concept was supported only by in vitro studies and does not relate to data on surveillance of antibiotic resistance (6, 21). Moreover, recent studies have demonstrated that development of chromosomal resistance to FOF entails a biological cost that reduces the virulence and biological fitness of pathogens (1, 10, 11, 22). In the present study, no development of increased resistance could be detected in samples of S. aureus recovered after FOF treatment. Considering the relatively long treatment period of 4 weeks, our data support the observation that development of clinically relevant FOF-resistant strains in vivo is rare.

In summary, our study has shown that FOF monotherapy is efficacious in the treatment of experimental MRSA osteomyelitis in rats. Although the study was limited in that no comparator drug was used, the rationale was to evaluate the efficacy of FOF monotherapy in terms of reduction of bacterial counts in osseous tissue and the development of resistance in vivo.

Considering the potent antimicrobial activity of FOF against multiresistant Gram-positive pathogens, including those with reduced susceptibility to glycopeptides, the finding of this study justifies the evaluation of FOF monotherapy in the treatment of osteomyelitis in humans.