1 Department of Orthopaedic Surgery, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam; 2 Department of Oral Cell Biology, Academic Center for Dentistry Amsterdam (ACTA); 3 Department of Oral Biochemistry, ACTA, Universiteit van Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands
Received 14 May 2004; returned 1 July 2004; revised 22 August 2004; accepted 27 August 2004
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Abstract |
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Methods: Dhvar-5 (12 mg or 24 mg/rabbit) was incorporated into polymethyl methacrylate (PMMA) beads as a local drug delivery system. For comparison, plain beads (control) and beads containing gentamicin as a sulphate (10 mg or 24 mg per rabbit) were also prepared. The beads were inserted into the inoculated femoral cavity of 36 rabbits, and 1 week later they were killed. The presence and severity of MRSA osteomyelitis was assessed by culture and histology.
Results: Both the 24 mg Dhvar-5 beads and the 24 mg gentamicin sulphate beads significantly reduced the bacterial load of the inoculated femora compared with the control chain. Although a 24 mg Dhvar-5 dose inhibited MRSA growth, it did not completely sterilize the femora. Sterilization occurred only in some of the gentamicin-treated specimens.
Conclusion: We conclude that both the gentamicin beads and the Dhvar-5 beads were only partially effective at preventing MRSA infection in this model.
Keywords: antimicrobial peptides , drug delivery , rabbits , PMMA beads
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Introduction |
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Antimicrobial peptides (AMPs) are a new and promising class of antibiotics, derived from naturally occurring peptides.7 These peptides are found on epithelial surfaces, in secretion fluids and in neutrophils, and thus form a first line of host defence as part of the innate immune system.8 AMPs have generated a lot of interest as possible new antimicrobial agents; van't Hof et al.9 have reviewed several AMPs under investigation. An advantage is that they have a diminished tendency to induce resistance because of the evolutionary difficulty in changing bacterial membrane structure.7,8
Dhvar-5 is an experimental AMP based on histatin-5, which is an antifungal peptide found in human saliva.10 Dhvar-5 has been designed to create a net positive charge at the C-terminus and a hydrophobic N-terminus (LLLFLLKKRKKRKY).10 The positive charge is presumed to play a crucial part in its antimicrobial activity.11 It disrupts and penetrates the negatively charged bacterial cell wall, and after cellular entrance it is targeted to intracellular organelles.12 The Dhvar-5 peptide has fungicidal and bactericidal activity and has antimicrobial activity against MRSA in vitro.13,14
In the light of antimicrobial resistance and the urgent need for new antimicrobial agents, we previously investigated the suitability of PMMA mini-beads as a carrier of Dhvar-5.15 These beads showed a high and prolonged in vitro release of Dhvar-5 in its biologically active form.15 Therefore, the aim of the present investigation was to evaluate the efficacy of Dhvar-5 and gentamicin beads in an in vivo MRSA osteomyelitis prevention model.
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Materials and methods |
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The femoral cavity of 36 rabbits was inoculated with approximately 107 colony-forming units (cfu) of MRSA in a modified prophylactic osteomyelitis model described by Nijhof et al.16 Immediately after inoculation, a custom-made PMMA mini-bead chain was inserted into the femur. The chains were prepared as described previously;15 the control group (n=8) received a chain of 10 plain cement beads (Osteopal; Biomet Merck, Darmstadt, Germany) without antibiotic. The AMP-treated animals received a chain of 10 beads containing a total of 12 mg (n=7) and 24 mg (n=9) of Dhvar-5. The gentamicin-treated animals received a chain prepared from Osteopal G (Biomet Merck) containing a total of either 10 mg (n=7) or 24 mg (n=5) of gentamicin sulphate. Experimental groups were augmented to contain at least seven animals for statistical purposes, except for the 12 mg Dhvar-5 group and the 24 mg gentamicin sulphate group. After 7 days, the animals were killed and the femoral cortex was cultured for the presence of MRSA and the number of cfu per gram of bone tissue was determined as a measure for treatment outcome.
Animals
Female New Zealand White (NZW) rabbits (3.54 kg) were obtained 3 weeks before surgery and allowed to acclimatize to the Clinical Animal Laboratory, Vrije Universiteit Medical Center (VUmc). The animals were housed in groups and allowed water and antibiotic-free rabbit diet ad libitum. The experimental protocol was approved by the Animal Ethics Committee of our institution.
Preparation of PMMA mini-beads
The bead chains were prepared as described previously.15 In short, two different quantities of Dhvar-5 (50 mg and 100 mg/g of cement powder) were mixed with the cement powder. The liquid monomer was added in a 1:2 liquid to powder ratio and the resulting cement paste was injected into a custom-made mould. The cement was allowed to polymerize on a stainless-steel wire (0.3 mm diameter), removed from the mould and stored inside a sealed tube at 20°C until use. This process produced a chain of 10 beads containing a total of either 12 or 24 mg of Dhvar-5. The 10 mg gentamicin sulphate chains were obtained by injecting the commercially available Osteopal G (Biomet Merck) bone cement into the mould. According to the manufacturer, Osteopal G is identical to Osteopal except for the gentamicin content. The gentamicin sulphate content of the Osteopal G cement powder was increased from 41.75 to 100 mg gentamicin sulphate per gram cement powder to produce the 24 mg gentamicin sulphate chains. From here on, the group dosages referred to as gentamicin are the gentamicin sulphate dosages. As each animal received one chain of 10 beads during surgery, the dosages mentioned are the amount of antibiotics incorporated into a chain of 10 beads, and therefore the amount per animal.
Bacterial strain
The MRSA strain (ATCC: BAA-811) was isolated from an osteomyelitis patient and was resistant to several antibiotics including gentamicin (Table 1). An overnight culture was grown in brain heart infusion (BHI) broth and washed in PBS. Subsequently, aliquots were prepared and stored at 80°C. The amounts of viable cfu were determined in defrosted aliquots by serial dilution and plating on blood agar. On the day of surgery a fresh aliquot was allowed to thaw and a bacterial suspension of 108 cfu/mL was prepared in PBS. The femoral canal was inoculated by a direct injection of 0.1 mL of bacterial suspension (107 cfu). After surgery the inoculum was serially diluted and plated on blood agar for determination of viable cfu count.
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The animals were weighed and subcutaneously injected with xylazine 2.5 mg/kg (Xylalin; CEVA Sante Animale, Maassluis, The Netherlands) and ketamine 62.5 mg/kg (Aescoket; Aesculaap, Boxtel, The Netherlands). Once sedated, the right hip area was shaved and painted with iodine. Subsequently, lidocaine 5 mg/kg (Pharmacy VUmc, Amsterdam, The Netherlands) was injected around the greater trochanter. Preoperative blood samples were taken from the right auricular vein for the determination of peripheral white blood cell count (WBC) and erythrocyte sedimentation rate (ESR). The animals were placed on their left side and the skin was again painted with iodine and the surgical area was isolated with sterile drapes. During surgery, the anaesthesia was maintained by free flow inhalation of a gas mixture containing 45% oxygen, 53.5% air and 1.5% isoflurane (Forene; Abbot BV, Hoofddorp, The Netherlands).
A 3 cm skin incision was made parallel to the femur shaft over the trochanter major of the right femur. After splitting the fascia and retracting the femoral biceps, the trochanter was exposed and the bone was notched to mark the drill site. The cortex was penetrated with a small drill (2.0 mm diameter), which was gradually increased to 3.5 mm diameter. The femoral canal was reamed with small stainless-steel brushes (Abrasives Center, Maastricht, The Netherlands) increasing in size up to 4.0 mm diameter. Subsequently the canal was flushed with 50 mL of 0.9% NaCl solution to remove any bony debris created by the reaming procedure. A small suction cuvette was inserted into the medullary canal to remove as much blood and NaCl solution as possible. In quick succession, the bacterial inoculation (0.1 mL) was injected and the beads inserted (Figure 1). The fascia and skin were closed with Vicryl 4/0 as soon as possible to prevent leakage of the inoculum. Post-operative pain relief was provided by injection of 0.15 mg of buprenorphine subcutaneously, which was repeated if necessary.
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After 7 days, the animals were euthanized, first by sedation with a subcutaneous injection of 2.5 mg/kg xylazine and 62.5 mg/kg ketamine, and then by killing with an intravenous injection of 75 mg/kg pentobarbital (Nembutal; CEVA Sante Animale, Naaldwijk, The Netherlands). Blood samples were drawn for WBC and ESR determination before administration of the pentobarbital. The right and left (control) femur were excised under strict aseptic conditions. After excision, radiographs of all femora were taken to document possible signs of osteomyelitis. Then a transverse saw cut was made in the middle of the femur, care being taken not to damage the bead chain inside the femoral canal. The bead chain was then gently removed from the femoral cavity. Subsequently, transverse sections were sawn from all proximal femora, for histological and bacteriological analysis (Figure 1).
Bacterial cultures
A transverse section of approximately 1 g from the proximal half of the femur was weighed and then homogenized in 50 mL of PBS at 15 000 rpm for 2 min (Sorval Omnimix; Dupont Instruments, Newton, CT, USA). A serial dilution was plated on blood agar and incubated for 24 h at 37°C. The bacterial load was expressed as cfu per gram of bone tissue. The detection limit of this method was approximately 2.5 x 103 cfu/g of bone depending on the mass of the specific homogenized bone section. For statistical analysis, the samples with no growth of bacteria in the quantitative assay were considered to have the detection limit value (2.5 x 103 cfu/g of bone) of bacterial load. Additionally, in order to assess the possible presence of MRSA below the detection limit of the quantitative method, a 100 µL sample of bone homogenate was cultured in BHI broth for at least 48 h at 37°C.
Histology
Bone samples were fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) and stored for at least 24 h at ±4°C, then thoroughly washed in PBS, dehydrated in an ascending alcohol series and then embedded in polymethyl methacrylate without plastoid.17 Undecalcified 30 µm thick sections were made with a 300 µm thick diamond-edge saw blade,18 and stained with a mixture of fuchsin and Methylene Blue.
Disease severity was assessed by a modified Smeltzer score.19 Disease severity is scored on the basis of periosteal inflammation, acute intra-medullar inflammation, chronic intra-medullar inflammation, and bone necrosis. However, as the complete histopathology normally takes more time to manifest itself than 7 days, only early signs of infection were scored, in particular, the presence of a periosteal reaction and necrotic bone.
Statistical analysis
The results of bacterial cultures were analysed using the Statistical Package for the Social Sciences (SPSS) version 10.1 for Windows. The Student's t-test for independent samples was used to analyse the quantitative microbiological data. The paired t-test was used for the analysis of body weight, temperature, WBC and ESR changes, and for analysis of these parameters between groups, one-way analysis of variance (ANOVA) was used. Furthermore, one-sided tests were carried out for comparison of each antibiotic group with the sham-treated control group, and two-sided tests were carried out for the comparison of Dhvar-5 and gentamicin groups. Differences were considered significant at P 0.05.
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Results |
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All animals tolerated and recovered well from surgery. However, one animal from the 12 mg Dhvar-5 group and two from the 24 mg Dhvar-5 group sustained a peroperative trochanter fracture and were thus excluded from the study. Also, one control rabbit was excluded due to a superficial infection. In total, 32 rabbits were included for statistical analysis.
There were no significant differences in weight or in temperature between the different groups (Figure 2a and b). Within groups, the 12 mg Dhvar-5 group lost 3.8% of its body weight (P=0.005), and the control group showed a minor increase in temperature of 0.5°C (P=0.05); no other significant differences were found.
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Microbiology
Quantitative data of the cultures from the femora are shown in Figure 3. In the sham-treated control group a mean (±S.E.M.) of 1.9 x 106 (±0.8 x 106) cfu/g of bone (n=7) were cultured and in the 12 mg (n=7) and 24 mg (n=7) Dhvar-5 groups, 0.78 x 106 (±0.2 x 106) and 0.51 x 106 (±0.1 x 106) cfu/g of bone were cultured, respectively. The difference between the control group and the 24 mg Dhvar-5 group was significant (P=0.04); between the 12 mg Dhvar-5 group and the control group the difference almost reached significance (P=0.07). Furthermore, in the 10 and 24 mg gentamicin groups means of 0.55 x 106 (±0.4 x 106) cfu/g of bone and 0.09 x 106 (±0.08 x 106) cfu/g of bone were cultured, respectively. The difference between the control group and the 10 mg gentamicin group almost reached significance (P=0.06), while the 24 mg gentamicin group had a significantly lower colony count compared with the control group (P=0.02) and compared with the 24 mg Dhvar-5 group (P=0.04). All left femora (not operated control) did not show any growth of bacteria.
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Table 2 also presents the histopathological results. In control animals, minor periosteal reaction (Figure 4a and b) was seen in all animals (7/7) and femoral necrosis in 6/7 animals. Similar results were seen in the 12 mg and 24 mg Dhvar-5 groups with a minor periosteal reaction in 6/6 and 6/7 animals, respectively. Bone necrosis was found in all but one of the 24 mg Dhvar-5 treated group. One animal of the 24 mg Dhvar-5 group had no signs of osteomyelitis. In the 10 mg gentamicin group, 5/7 animals showed a periosteal reaction and 7/7 showed bone necrosis. In the 24 mg gentamicin group, 3/5 animals showed early signs of osteomyelitis. The histopathological scores revealed no significant differences between the groups (data not shown).
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Discussion |
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An advantage of this model is the minor systemic signs of infection (Figure 2a and b). Despite the relatively large inoculum (107 cfu), none of the operated animals developed sepsis. This was also reflected in the systemic infection parameters, which showed only minor changes in all groups (Figure 2c and d). Another advantage of this study is the good and reproducible distribution of PMMA beads throughout the femoral canal (Figure 1), the number and proximity of antibiotic beads is well known to influence antibiotic tissue levels in the bone.2 Local treatment of osteomyelitis consists of debriding necrotic bone and inserting as much as possible antibiotic beads into the medullary canal, adding to the clinical relevance of this model.1
The data presented show that 24 mg of Dhvar-5 incorporated in custom-made PMMA mini-beads can inhibit MRSA proliferation in vivo, significantly reducing bacterial load compared with controls (P=0.04) (Figure 3). The 12 mg group also demonstrated a trend (P=0.07) towards reduction in bacterial load per gram of bone. The data therefore suggest a doseresponse effect of Dhvar-5 in vivo (Figure 3). Still, even a high dose of Dhvar-5 was not able to eradicate all bacteria and sterilize the femur. The 24 mg gentamicin group on the other hand showed inhibition of MRSA proliferation (P=0.02) and sterilized three out of five femora (Table 2 and Figure 3). The low-dose gentamicin (10 mg) group showed a trend (P=0.06) towards reducing the number of cfu per gram of bone and sterilized three out of seven femora.
The MRSA strain was highly resistant to several antibiotics including gentamicin (MIC = 256 mg/L, Table 1). Despite this marked resistance, the high-dose gentamicin beads did prevent infection in three of five rabbits. Several studies have shown that the local application of gentamicin in PMMA beads can result in local bone tissue levels around this high MIC value,2,22 thus explaining the ability of the gentamicin beads to sterilize some of the femurs in this study.
There are numerous reports indicating an increase in antimicrobial resistance, in particular of S. aureus and coagulase-negative staphylococci (CoNS).3,4,2325 In addition to the high prevalence of MRSA, the first strains of vancomycin-resistant S. aureus (VRSA) are now being reported.6 Moreover, the development of antimicrobial resistance is increased in biofilms.26 The formation of biofilms renders bacteria very resistant to conventional antibiotics and can even result in bacteria colonizing gentamicin-containing PMMA beads.5 These developments have increased concern over the future applicability of conventional antibiotics, and spurred the development of novel antimicrobial agents.7,8,2734
Over the past decade, numerous naturally occurring AMPs have been identified and their pivotal role in the mechanism of our innate immune system has been discovered.7,8 Several research groups have specifically described options for the development of AMPs as new antimicrobial agents.7,8,2734 The rBPI21 antimicrobial peptide has reached Phase III clinical trials and has shown promising clinical results: improving the outcome of meningococcal sepsis in children.28
Based on our in vitro release study, we first chose to incorporate 12 and 24 mg of Dhvar-5 in the PMMA beads,15 as this should have resulted in a continuous release of a sufficient quantity of Dhvar-5 to kill bacteria at the inoculated site for the duration of the experiment. After 7 days of release in vitro, 65% of Dhvar-5 is released from the 24 mg chain as opposed to 37% of gentamicin from the 24 mg gentamicin beads (ref. 15; and C. Faber, R. J. W. Hoogendoorn, D. M. Lyaruu, H. P. Stallmann, J. van Marle, A. V. Nieuw Amerongen, T. H. Smit & P. I. J. M. Wuisman, unpublished results). The high dose of Dhvar-5 resulted in a significantly reduced bacterial load per gram of bone (Figure 3). However, as there was no method available to measure in vivo concentrations of Dhvar-5, we could not determine the systemic or local concentrations of Dhvar-5. It is therefore difficult to relate the very high release characteristics in vitro of Dhvar-5 to the results obtained in vivo. There are no data available on the in vivo half-life of Dhvar-5. Other AMPs are known to have a much shorter half-life than gentamicin.29 It is therefore possible that Dhvar-5 is cleared more rapidly than gentamicin. Also, in vitro studies have shown that Dhvar-5 can be degraded by proteolytic enzymes such as trypsin.35 This could possibly reduce local tissue levels of Dhvar-5 in vivo. Furthermore, some AMPs have been reported to bind to serum proteins, resulting in reduced antimicrobial activity.36 In this study, a combinationz of these factors could have resulted in lower local free and/or active tissue concentrations of Dhvar-5, possibly compromising the in vivo efficacy, thus explaining the relatively better performance of the gentamicin beads compared with the Dhvar-5 beads, despite the marked gentamicin resistance of the MRSA strain.
This study demonstrates the in vivo antimicrobial activity of Dhvar-5 against MRSA. However, the gentamicin beads demonstrated the best efficacy in preventing MRSA osteomyelitis in this study. Future development of Dhvar-5 and other AMPs as novel antimicrobial agents is largely dependent on the ability to control aspects such as the pharmacodynamics, inhibition of enzymic proteolysis and prevention of possible inactivation by binding to proteins. Numerous opportunities can be found in changing amino acid sequences whilst retaining or even improving the antimicrobial activity. This research provides a basis for the further development of AMPs as an alternative for conventional antibiotic agents for the treatment of drug-resistant pathogens.
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Acknowledgements |
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Supplementary data
Supplementary data accompanying this paper are available at JAC Online.
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Footnotes |
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References |
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2 . Wahlig, H., Dingeldein, E., Bergmann, R. et al. (1978). The release of gentamicin from polymethylmethacrylate beads. An experimental and pharmacokinetic study. Journal of Bone and Joint Surgery. British volume 60-B, 2705.[Medline]
3
.
Witte, W. (1999). Antibiotic resistance in Gram-positive bacteria: epidemiological aspects. Journal of Antimicrobial Chemotherapy 44, Suppl. A, 19.
4
.
Kac, G., Buu-Hoi, A., Herisson, E. et al. (2000). Methicillin-resistant Staphylococcus aureus. Nosocomial acquisition and carrier state in a wound care center. Archives of Dermatology 136, 7359.
5
.
Neut, D., van de Belt, H., Stokroos, I. et al. (2001). Biomaterial-associated infection of gentamicin-loaded PMMA beads in orthopaedic revision surgery. Journal of Antimicrobial Chemotherapy 47, 88591.
6 . Whitener, C. J., Park, S. Y., Browne, F. A. et al. (2004). Vancomycin-resistant Staphylococcus aureus in the absence of vancomycin exposure. Clinical Infectious Diseases 38, 104955.[CrossRef][ISI][Medline]
7 . Hancock, R. E. (1997). Peptide antibiotics. Lancet 349, 41822.[CrossRef][ISI][Medline]
8 . Zasloff, M. (2002). Antimicrobial peptides of multicellular organisms. Nature 415, 38995.[CrossRef][ISI][Medline]
9 . van 't Hof, W., Veerman, E. C., Helmerhorst, E. J. et al. (2001). Antimicrobial peptides: properties and applicability. Journal of Biological Chemistry 382, 597619.[CrossRef]
10 . Helmerhorst, E. J., Reijnders, I. M., van 't Hof, W. et al. (1999). A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides. FEBS Letters 449, 10510.[CrossRef][ISI][Medline]
11 . Helmerhorst, E. J., van 't Hof, W., Veerman, E. C. et al. (1997). Synthetic histatin analogues with broad-spectrum antimicrobial activity. Biochemical Journal 326, 3945.[ISI][Medline]
12
.
Helmerhorst, E. J., Breeuwer, P., van 't Hof, W. et al. (1999). The cellular target of histatin 5 on Candida albicans is the energized mitochondrion. Journal of Biological Chemistry 274, 728691.
13 . Ruissen, A. L., Groenink, J., Helmerhorst, E. J. et al. (2001). Effects of histatin 5 and derived peptides on Candida albicans. Biochemical Journal 356, 3618.[CrossRef][ISI][Medline]
14 . Lyaruu, D., van 't Hof, W. & Veerman, E. C. (2000). Anti-microbial activity of human saliva histatin analogues against methicillin-resistant S. aureus (MRSA). Journal of Dental Research 79, 227.
15
.
Faber, C., Stallmann, H. P., Lyaruu, D. M. et al. (2003). Release of antimicrobial peptide Dhvar-5 from polymethylmethacrylate beads. Journal of Antimicrobial Chemotherapy 51, 135964.
16 . Nijhof, M. W., Dhert, W. J., Fleer, A. et al. (2000). Prophylaxis of implant-related staphylococcal infections using tobramycin-containing bone cement. Journal of Biomedical Materials Research 52, 75461.[CrossRef][ISI][Medline]
17 . Theuns, H. M., Bekker, H., Fokkenrood, H. et al. (1993). Methyl-methacrylate embedding of undecalcified rat bone and simultaneous staining for alkaline and tartrate resistant acid phosphatase. Bone 14, 54550.[ISI][Medline]
18 . Klein, C. P., Sauren, Y. M., Modderman, W. E. et al. (1994). A new saw technique improves preparation of bone sections for light and electron microscopy. Journal of Applied Biomaterials 5, 36973.[ISI][Medline]
19 . Smeltzer, M. S., Thomas, J. R., Hickmon, S. G. et al. (1997). Characterization of a rabbit model of staphylococcal osteomyelitis. Journal of Orthopaedic Research 15, 41421.[ISI]
20 . Nijhof, M. W., Stallmann, H. P., Vogely, H. C. et al. (2000). Prevention of infection with tobramycin-containing bone cement or systemic cefazolin in an animal model. Journal of Biomedical Materials Research 52, 70915.[CrossRef][ISI][Medline]
21 . Norden, C. W., Myerowitz, R. L. & Keleti, E. (1980). Experimental osteomyelitis due to Staphylococcus aureus or Pseudomonas aeruginosa: a radiographic-pathological correlative analysis. British Journal of Experimental Pathology 61, 45160.[ISI][Medline]
22 . Harle, A. & Ritzerfeld, W. (1979). The release of gentamicin into the wound secretions from polymethylmethacrylate beads. A study with reference to the animal experiment. Archives of Orthopaedic Trauma Surgery 95, 6570.[CrossRef][ISI]
23
.
Denis, O., Nonhoff, C., Byl, B. et al. (2002). Emergence of vancomycin-intermediate Staphylococcus aureus in a Belgian hospital: microbiological and clinical features. Journal of Antimicrobial Chemotherapy 50, 38391.
24 . Hornberg, C., Schafer, T. R., Koller, A. et al. (2003). The MRSA patient in technical orthopaedics and rehabilitation. Orthopäde 32, 2137.[ISI][Medline]
25 . Hornberg, C., Koller, A., Buhring, W. et al. (2001). Methicillin-resistant Staphylococcus aureus (MRSA). Current status and significance of preventing infection in technical orthopedics. Orthopäde 30, 2315.[ISI][Medline]
26 . Stewart, P. S. & Costerton, J. W. (2001). Antibiotic resistance of bacteria in biofilms. Lancet 358, 1358.[CrossRef][ISI][Medline]
27 . Groenink, J., Walgreen-Weterings, E., van 't Hof, W. et al. (1999). Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens. FEMS Microbiology Letters 179, 21722.[CrossRef][ISI][Medline]
28 . Levin, M., Quint, P. A., Goldstein, B. et al. (2000). Recombinant bactericidal/permeability-increasing protein (rBPI21) as adjunctive treatment for children with severe meningococcal sepsis: a randomised trial. rBPI21 Meningococcal Sepsis Study Group. Lancet 356, 9617.[CrossRef][ISI][Medline]
29
.
Nibbering, P. H., Ravensbergen, E., Welling, M. M. et al. (2001). Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria. Infection and Immunity 69, 146976.
30
.
Navon-Venezia, S., Feder, R., Gaidukov, L. et al. (2002). Antibacterial properties of dermaseptin S4 derivatives with in vivo activity. Antimicrobial Agents and Chemotherapy 46, 68994.
31
.
Steinstraesser, L., Tack, B. F., Waring, A. J. et al. (2002). Activity of novispirin G10 against Pseudomonas aeruginosa in vitro and in infected burns. Antimicrobial Agents and Chemotherapy 46, 183744.
32
.
Giacometti, A., Cirioni, O., Ghiselli, R. et al. (2002). Potential therapeutic role of cationic peptides in three experimental models of septic shock. Antimicrobial Agents and Chemotherapy 46, 21326.
33
.
Giacometti, A., Cirioni, O., Gov, Y. et al. (2003). RNA III inhibiting peptide inhibits in vivo biofilm formation by drug-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 47, 197983.
34
.
Giacometti, A., Cirioni, O., Ghiselli, R. et al. (2004). Cathelicidin peptide sheep myeloid antimicrobial peptide-29 prevents endotoxin-induced mortality in rat models of septic shock. American Journal of Respiratory and Critical Care Medicine 169, 18794.
35 . Den Hertog, A. L., Wong Fong Sang, H. W., Kraayenhof, R. et al. (2004). Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization. Biochemical Journal 379, 66572.[CrossRef][ISI][Medline]
36 . Wang, Y., Johansson, J., Agerberth, B. et al. (2004). The antimicrobial peptide LL-37 binds to the human plasma protein apolipoprotein A-I. Rapid Communications in Mass Spectrometry 18, 5889.[CrossRef][ISI][Medline]
37 . Lorain, V. (1996). Antibiotics in Laboratory Medicine. Williams & Wilkins, Baltimore, MD, USA.
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