1 Institute of Infectious Diseases and Public Health, University of Ancona, Ancona; 2 Department of General Surgery, I.N.R.C.A. I.R.C.C.S., University of Ancona, Ancona; 3 Biotechnology Centre, Research Department, I.N.R.C.A. I.R.R.C.S., Ancona, Italy
Received 27 March 2003; returned 21 May 2003; revised 20 June 2003; accepted 29 June 2003
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Abstract |
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Methods: Graft infections were established in the subcutaneous tissue of adult male Wistar rats by implantation of Dacron prostheses, followed by topical inoculation with S. epidermidis. The study comprised: one group without inoculation; one inoculated group without prophylaxis; six inoculated groups that received intraperitoneal linezolid (8 mg/kg), levofloxacin (7 mg/kg) or vancomycin (7 mg/kg) alone or in combination at the dosages mentioned above. Each group included 20 animals. The grafts were removed after 7 days and evaluated by quantitative culture.
Results: Quantitative graft cultures from animals treated with a single drug showed a significant efficacy only for linezolid. The efficacy of levofloxacin was similar to that of vancomycin. Combination studies demonstrated that only the treatments that included linezolid produced no evidence of staphylococcal infection.
Conclusions: Linezolid as perioperative prophylaxis can be useful for the prevention of graft infections caused by multiresistant staphylococcal strains.
Keywords: antibiotic prophylaxis, oxazolidinones, vascular surgery, GISE
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Introduction |
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CoNS, chiefly the skin commensal Staphylococcus epidermidis, are among the most common pathogens to cause graft infection, a severe complication of vascular reconstructions.2 Most important in the pathogenesis of foreign-body-associated infection caused by staphylococci is the colonization of the polymer surface. Bacteria that adhere to implanted medical devices can encase themselves in a hydrated matrix of polysaccharide and protein, and form a slimy layer known as a biofilm. Formation of these sessile communities and their inherent resistance to antimicrobial agents are the root of many persistent and chronic bacterial infections.3,4
Oxazolidinones are a new class of antimicrobials with a unique mechanism of action. They inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit; this binding prevents formation of a functional initiation complex in bacterial translation systems. Linezolid has been approved by the Food and Drug Administration for use in treating infections caused by Gram-positive organisms, including multidrug-resistant isolates of staphylococci, streptococci and enterococci.5
Levofloxacin, a fluoroquinolone with somewhat enhanced activity against Gram-positive cocci, is the L-stereoisomer of ofloxacin, which is a racemic mixture that contains equal parts of the L- and D-stereoisomers.6
In the 1950s, vancomycin was the first glycopeptide to be introduced into clinical practice, and for almost three decades following its introduction, resistance was reported only rarely and appeared to have little clinical significance. In the 1980s, however, vancomycin resistance emerged in CoNS, especially S. epidermidis, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus haemolyticus and Staphylococcus xylosus.7
Linezolid was introduced into clinical practice as a consequence of the emergence of multidrug-resistant staphylococci. We have used a rat model to investigate its efficacy in the prevention of graft infections, following the development of a strain with intermediate resistance to vancomycin.
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Materials and methods |
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VISE (vancomycin-intermediate S. epidermidis) is the acronym used to describe the strain of S. epidermidis with intermediate resistance to vancomycin. Commercially available S. epidermidis ATCC 12228 was used as the quality control strain in the in vitro investigations.
Drugs
Linezolid was obtained from Pharmacia & Upjohn, Kalamazoo, MI, USA. Vancomycin was obtained from Sigma-Aldrich, Milan, Italy. Levofloxacin was obtained from GlaxoSmithKline, Verona, Italy. Powders were diluted in accordance with manufacturers recommendations. Solutions of drugs were made fresh on the day of assay.
Antimicrobial susceptibility testing
The antimicrobial susceptibilities of the strains were determined by the broth microdilution method described by the NCCLS. In addition, the NCCLS reference disc diffusion method, which utilizes discs of vancomycin 30 µg, was used to test the strain for susceptibility to vancomycin.8,9 Experiments were performed in triplicate.
In vivo rat model
Graft infections were established in the subcutaneous tissue of 160 adult male Wistar rats by implanting Dacron prostheses, followed by topical inoculation of the VISE strain. The study included one control group without inoculation (C1); one inoculated control group without prophylaxis (C2); and six inoculated groups that received intraperitoneal linezolid (8 mg/kg), levofloxacin (7 mg/kg) or vancomycin (7 mg/kg), alone or in combination, at the dosages given above, 30 min before implantation. Each group included 20 animals. Rats were anaesthetized with ether, the hair on the back was shaved and the skin cleaned with 10% povidoneiodine solution. A 1.5 cm incision was made on each side of the median line to form a subcutaneous pocket. Aseptically, 1 cm2, sterile, collagen-sealed, double velour knitted, polyethylene terephthalate (Dacron) grafts (Albograft, Sorin Biomedica Cardio, Saluggia VC, Italy) were implanted into the pockets. The pockets were closed by means of skin clips, and physiological solution (1 mL) containing the VISE strain at a concentration of 2 x 107 cfu/mL was inoculated onto the graft surface. The animals were returned to individual cages and examined thoroughly daily. Toxicity was evaluated on the basis of the presence of local signs of perigraft inflammation, anorexia, weight loss, vomiting, diarrhoea, fever and behavioural alterations. All grafts were explanted 7 days after implantation. The study was approved by the Animal Research Ethics Committee of the I.N.R.C.A. I.R.R.C.S., University of Ancona.
Serum antibiotic concentration measurements and kinetics
Preventive experiments were performed to measure serum linezolid, levofloxacin and vancomycin levels in uninfected animals receiving intraperitoneal drugs. Blood samples were obtained from the tail vein of 18 rats (six rats for each agent) 1, 2 and 4 h after a single intraperitoneal dose of linezolid (8 mg/kg), levofloxacin (7 mg/kg) or vancomycin (7 mg/kg). Drug levels were measured by bioassay: a spore suspension of Bacillus subtilis ATCC 6633 suspended in tryptic soy agar was used. The plates were read after incubation at 30°C for 18 h.
Assessment of the infection
The explanted grafts were placed in sterile tubes, washed in sterile saline solution, placed in tubes containing 10 mL of phosphate-buffered saline solution and sonicated for 2 min to remove the adherent bacteria from the grafts. Quantification of viable bacteria was performed by culturing serial 10-fold dilutions (0.1 mL) of the bacterial suspension on blood agar plates. All plates were incubated at 37°C for 48 h and evaluated for the presence of the VISE strain. The organisms were quantified by counting the number of cfu per plate. The limit of detection for this method was 10 cfu/mL.
Statistical analysis
MIC values are presented as the mode of three separate experiments. Quantitative culture results from all groups are presented as mean ± S.D., and the statistical comparisons between groups were made using analysis of variance (ANOVA) on the log-transformed data. Significance was accepted when the P value was 0.05.
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Results and discussion |
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According to the broth microdilution method, the clinical isolate showed intermediate resistance to vancomycin. Vancomycin exhibited MICs of 0.25 and 8 mg/L for S. epidermidis ATCC 12228 and the VISE strain, respectively. The different pattern of susceptibility was confirmed by the disc diffusion test: S. epidermidis ATCC 12228 showed a zone size of 18 mm for vancomycin, whereas the intermediate resistance of the VISE strain to the glycopeptide was demonstrated by a zone size of 11 mm. The two organisms were similarly susceptible to linezolid, which showed MICs of 1.00 and 2.00 mg/L for S. epidermidis ATCC 12228 and the VISE strain, respectively. Finally, different activity was exerted by levofloxacin, which showed MICs of 0.5 and 16 mg/L for the control and the resistant strain, respectively.
In vivo experiments
All rats included in the untreated control group C2 demonstrated evidence of graft infection, whereas none of the animals included in the uninoculated control group C1 had microbiological evidence of graft infection. Among the animals that received single antibiotic prophylaxis, only linezolid demonstrated significant efficacy compared with the inoculated control group (P < 0.001). There were no significant differences between levofloxacin and vancomycin (Table 1).
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After a single intraperitoneal injection, linezolid, levofloxacin and vancomycin reached peak levels of 17.4 mg/L, 4.9 mg/L and 13.2 mg/L, respectively. Finally, none of the animals included in any group died or had clinical evidence of drug-related adverse effects, such as local signs of perigraft inflammation, anorexia, vomiting, diarrhoea or alterations in behaviour.
The emergence of antibiotic-resistant Gram-positive cocci has created a pressing need for effective alternative antibiotics.1,2,7 The new oxazolidinone, linezolid, has demonstrated a selective spectrum of antibacterial activity, primarily against Gram-positive aerobic bacteria, although bacterial resistance to linezolid has been reported recently.10 It has been assessed mainly in patients with glycopeptide-resistant Gram-positive infections.5
Our results demonstrated that the use of linezolid alone, or combined with other drugs, can result in significant inhibition of the bacterial growth of the resistant strain, even with high concentrations of resistant organisms inoculated topically on the Dacron prostheses. In our model, the explanted grafts had to be sonicated to remove the adherent bacteria. This method led us to obtain quantitative cultures, not only concerning the bacteria included on the Dacron, but also those grown on biofilm surrounding the grafts. However, the animal model in the present study is not directly comparable with graft implantation into a blood vessel, and caution is needed. These results need to be compared with the real situation of an implanted graft in the aorta of a living human being.
The clinical experience clearly shows that the prevention of serious staphylococcal infections, such as vascular graft infection, requires the use of antimicrobial agents with bactericidal activity. For this reason, prevention through effective antibiotic prophylaxis plays an important role in patient mortality and the cost-effectiveness of hospital care. In view of our results, further studies are needed to investigate the potential usefulness of linezolid in perioperative chemoprophylaxis of prosthetic surgery.
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Footnotes |
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References |
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Chambers, H. F., Xiang Liu, Q., Liuxin Chow, L. et al. (1999). Efficacy of levofloxacin for experimental aortic-valve endocarditis in rabbits infected with viridans group streptococcus or Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 43, 27426.
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10 . Gonzales, R. D., Schreckenberger, P. C., Graham, M. B. et al. (2001). Infections due to vancomycin-resistant Enterococcus faecium resistant to linezolid. Lancet 357, 1179.[CrossRef][ISI][Medline]