Release of Antibiotics from Polymethylmethacrylate Cement
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1 REPRINT Journal of Chemotherapy Vol n. 5 ( ) REVIEW Release of Antibiotics from Polymethylmethacrylate Cement E. BERTAZZONI MINELLI - C. CAVEIARI - A. BENINI Department of Medicine and Public Health, Unit of Pharmacology, University of Verona, Verona, Italy. Corresponding author: Prof. E. Bertazzoni Minelli, Department of Medicine and Public Health, Unit of Pharmacology, Policlinico G.B. Rossi, Verona, Italy. Tel.: / ; Fax: elisa.bertazzoni@univr.it Summary The increase in resistance rates to antibiotics of bacteria isolated from infected hip joints, particularly staphylococci, prompted us to investigate the usefulness of antibiotic combinations such as gentamicin plus vancomycin. Cylinder test specimens of polymethylmethacrylate (PMMA) cement (Cemex, Tecres) containing gentamicin alone, vancomycin alone and both drugs in combination, were studied. The antibiotic concentrations were determined using a microbiological method and fluorescence polarization immunoassay (FPIA). The release of gentamicin alone, vancomycin alone and in combination from PMMA cement was prompt. The combination revealed synergistic antimicrobial activity against Escherichia coli and Enterococcus faecalis. FPIA showed that gentamicin and vancomycin delivery rates from PMMA cement were different. Gentamicin alone and in combination with vancomycin presented similar release rates from PMMA cement (1.50%). Vancomycin release from PMMA cylinders impregnated with the combination was lower (0.51%) than that from cylinders with vancomycin alone (1.16%). Vancomycin showed a 34.1% loss of microbiological activity at 37 C after 10 days of incubation; the reduction corresponded to 15.0% when measured by FPIA. Results obtained with test specimens are indicative for the preparation of antibiotic-impregnated cements for different human prostheses. Key words: Gentamicin, vancomycin, combination, elution, FPIA, surgical infections, surgical cement, drug delivery system. INTRODUCTION Infection is a serious complication in orthopedic surgery, and delivery of antibiotics to the surgical area is an effective way of both preventing and treating infections 1. Antibioticimpregnated polymethylmethacrylate (PMMA) cement was developed to treat local infections of bone and soft tissue 2. The increase in resistance to antibiotics of E.S.I.F.T. srl - Firenze ISSN X
2 RELEASE OF ANTIBIOTICS FROM POLYMETHYLMETHACRYLATE CEMENT 493 bacteria isolated from infected hip joints, particularly staphylococci 2,3, prompted us to investigate the usefulness of antibiotic combinations such as gentamicin plus vancomycin. The aim of this study was to determine: a) the release rates of gentamicin alone and vancomycin alone and in combination from PMMA cement test specimens, and b) the antimicrobial activity of the antibiotics and their combination after release from PMMA cement. MATERIALS AND METHODS Specimens Cylinders 6 mm in diameter and 11 mm in height, each weighing 400 mg, were prepared from the polymerized cement (Cemex, Tecres) with mold under aseptic conditions (sterile chamber). 2.5 g of gentamicin (powder, Eu. Ph. Grade) and 5 g of vancomycin (Vancocin powder, Eli Lilly, Milan, Italy) alone and in combination were mixed with 100 g of polymethylmethacrylate (PMMA) copolymer powder prior to the addition of liquid polymer (35 ml) under laminar flow. An exothermic polymerization reaction occurs yielding solidification over a period of 10 minutes. Each cylinder contained vancomycin (20 mg) and gentamicin (10 mg) alone and their combination (20 mg vancomycin + 10 mg gentamicin). We studied 4 cylinders with vancomycin alone, 4 with gentamicin alone and 4 with the combination. Cylinders were kindly prepared by Tecres S.p.A. (Verona, Italy) utilizing the same procedures as for commercial bone cement products (Cemex, Tecres). Polymerization reaction of PMMA with vancomycin is slower when compared to the same reaction in the presence of gentamicin; moreover, vancomycin mixes with PMMA with more difficulty than gentamicin. Elution of antibiotics The PMMA test specimens were immersed in tubes with a defined volume (2 ml) of phosphate buffer (PB, 0.2M, ph = 8.0, suitable for in vitro antibiotic stability assay) at 37 C for 10 days. PB was removed and replaced with the same volume of fresh buffer (2 ml) after 15 min, and 1, 2, 4, 24, 72 and 240 hours of immersion. The removed buffer was frozen at - 24 C. The elution samples from each test specimen were analyzed in duplicate in the same experiment. Recovery of antibiotic was calculated as total eluted micrograms. Standard concentrations of antibiotics were prepared in PB and processed along with samples. Different stock solutions of the combination were prepared by addition of (a) 1 part of gentamicin (160 mg/l) to 1 part of vancomycin (160 mg/l) and (b) 1 part of gentamicin (160 mg/l) to 2 parts of vancomycin (320 mg/l), then diluted 1:1 to obtain standard concentrations for determinations. Antibiotic concentrations in eluted samples and standards were analyzed in parallel with both methods, bioassay and immunoassay. Bioassay Antibiotic concentrations alone and in combination were determined by the standard large plate agar-well diffusion method 4 according to NCCLS guidelines 5. Briefly, Bacillus subtilis spore suspension ATCC 6633 (final concentration 0.02%) seeded in Isosensitest Agar (Oxoid Unipath Ltd, Basingstoke, England) and Enterococcus faecalis ATCC (final concentration of overnight culture 0.1%) in Antibiotic Medium 1 (Difco Laboratories, Detroit, MI) were used as test microorganisms to evaluate the concentrations of gentamicin, vancomycin and the gentamicin-vancomycin combination. Escherichia coli ATCC (final concentration of overnight culture, 0.1%) was also used as test microorganism in Isosensitest Agar to evaluate gentamicin and the combination concentrations. Inoculated agar (100 ml) was poured into sterile, level, squared plastic Corning bioassay dishes (245 by 245 mm, Corning Incorporated, USA). After solidification, 80-mm-diameter wells were punched out and filled with 50 µl of solution (samples and standards). After overnight incubation at 37 C, the diameters of the inhibition zones for each standard and eluted samples were measured. The unknown antibiotic concentrations in the samples were determined by standard curves created with known antibiotic concentrations and plotted on semilogarithmic scale (Log concentration vs zone of inhibition). All samples and standard concentrations were assayed in duplicate or triplicate.
3 494 E. BERTAZZONI MINELLI - C. CAVEIARI - A. BENINI Gentamicin assay was linear over a range of mg/l (R 2 = 0.98); mg/l (R 2 = 0.99); mg/l (R 2 = 0.98) against Bacillus subtilis, Escherichia coli and Enterococcus faecalis, respectively. Vancomycin assay was linear over a range of mg/l (R 2 = 0.98) and mg/l (R 2 = 0.97) against B. subtilis and E. faecalis, respectively. The combination (1:1 and 1:2) assay was linear over a range of mg/l (R 2 = 0.97); mg/l (R 2 = 0.97) and mg/l (R 2 =0.97) against B. subtilis, E. coli and E. faecalis, respectively. The between-day coefficients of variation were 6.5% and 12.8% for the highest and lowest gentamicin concentrations, respectively, against B. subtilis; 9.1% and 13.8% for the highest and lowest gentamicin concentrations, respectively, against E. coli; 9.3% and 10.9% for the highest and lowest gentamicin concentrations, respectively, against E. faecalis. The between-day coefficients of variation were 3.9% and 8.7% for the highest and lowest vancomycin concentrations, respectively against B. subtilis; 4.8% and 5.5% for the highest and lowest vancomycin concentrations, respectively, against E. faecalis. The between-day coefficients of variation were 6.6% and 5.9% for the highest and lowest gentamicin+vancomycin combination (1:1) concentrations, respectively, against B. subtilis; 4.4% and 1.8% for the highest and lowest gentamicin+vancomycin (1:1) combination concentrations, respectively, against E. faecalis; 10% and 16.7% for the highest and lowest concentrations of the combination against E. coli, respectively. The between-day coefficients of variation were 1.31% and 0% for the highest and lowest gentamicin+vancomycin combination (1:2) concentrations, respectively, against B. subtilis; 2.7% and 0.1% for the highest and lowest gentamicin+vancomycin combination (1:2) concentrations, respectively, against E. coli; 4.6% and 0.15% for the highest and lowest gentamicin+vancomycin combination (1:2) concentrations, respectively, against E. faecalis. Synergy testing Synergy testing was performed using the checkerboard method 6 in microtiter trays with Mueller-Hinton broth (Difco). Gentamicin and vancomycin were tested at seven concentrations (from 0.6 to 40.0 mg/l for gentamicin and from 5.0 to mg/l for vancomycin, respectively) against E. coli (final inoculum 1x10 5 CFU/ml) and E. faecalis (final inoculum 1 x10 5 CFU/ml). Fractional inhibitory concentration (FIC) indices were calculated and interpreted according to the method described by Eliopoulos and Moellering 6. FIC indices were interpreted as synergistic if the values were <0.5, indifferent or additive if the values were from 0.5 to 4.0 and antagonistic if the values were >4.0. Immunoassay In parallel, concentrations of gentamicin alone, vancomycin alone and the gentamicinvancomycin combination in eluted samples and standards were also determined by fluorescence polarization immunoassay (FPIA). Kits, calibrators, controls and buffers were purchased from an Italian supplier, calibrated and used according to the manufacturer s recommendations (TDx, Abbott) 7. The lowest measurable level of drug concentration was defined as that which could be distinguished from 0 with 95% confidence; this was determined as 0.27 mg/l for gentamicin and 2.0 mg/l for vancomycin 7. Tests were carried out in duplicate. Stability The stability of antibiotics alone and of the combination was evaluated in different conditions. Serial concentrations (from 1.25 to 20.0 mg/l) of each drug and the combination (1:1) were prepared in PB (2 ml volume per sample, in screw tubes), placed at 37 C and assayed at time 0 and after 3 and 10 days of incubation. In another set of experiments we evaluated the stability of antibiotics over time. The different concentrations of antibiotics, prepared as described above, were maintained at 4 C for 4 weeks. Both the microbiological method and FPIA were utilized to assay drug concentrations. Statistical analysis The results were analyzed statistically using Student s t-test.
4 RELEASE OF ANTIBIOTICS FROM POLYMETHYLMETHACRYLATE CEMENT 495 Antimicrobial activity RESULTS The release of gentamicin alone, vancomycin alone and the gentamicin-vancomycin combination from PMMA cement was immediate, presenting a peak after 15 minutes and high concentrations in the first 4 hours of elution. The release was constant and gradual over the following 10 days (Figure 1). elution. The combination revealed strong antimicrobial activity compared to that exerted by the antibiotics alone, with a positive effect. The antimicrobial activity of the combination would appear to be synergistic, as shown both by the antibacterial action against B. subtilis (highly susceptible), and by the good activity against E. faecalis. We registered the unexpected improvement in the activity of the combination against E. coli, a microorganism not susceptible to vancomycin, as is well known (Figure 2). Moreover, the microbiological assay of vancomycin in agar medium confirmed the poor capacity of diffusion of glycopeptides represented by small zone of inhibition even at high concentrations. FIGURE 2 - Amount of gentamicin, vancomycin and gentamicin + vancomycin combination eluted from cylinders showing antimicrobial activity against different test microorganisms (Bacillus subtilis spore suspension ATCC 6633, Enterococcus faecalis ATCC and Escherichia coli ATCC 25922). Values are expressed as µg (mean + SD) of antibiotic eluted at the beginning and at the end of the immersion period (total amount eluted during 10 days). FIGURE 1- Recovery of gentamicin alone, vancomycin alone and gentamicin-vancomycin combination at different elution times determined using a microbiological method (B. subtilis) (mean + SD). The concentrations of antibiotics eluted at different times showed effective antimicrobial activity both alone and in combination exceeding the MIC for E. coli and E. faecalis with different periods of activity (Figure 2). The concentrations of vancomycin alone were effective against E. faecalis for the first period of Synergism between gentamicin and vancomycin was also verified and confirmed by the checkerboard method. The MIC of gentamicin was 5 mg/l for E. coli and 40 mg/l for E. faecalis; the MIC of vancomycin was 2.5 mg/l for E. faecalis and >320 mg/l for E. coli. Gentamicin and vancomycin in combination exhibited a synergistic effect against E. coli and E. faecalis with FIC indices of and 0.365, respectively. Values <0.5 indicate a synergistic effect.
5 496 E. BERTAZZONI MINELLI - C. CAVEIARI - A. BENINI Immunoassay The FPIA method is able to measure vancomycin and gentamicin separately even when in combination. The concentrations of the two antibiotics alone as determined by FPIA were higher than those obtained with the microbiological method, as shown in Table 1. Gentamicin and vancomycin reached their peak immediately (after 15 min) maintaining good drug elution during the first 4 hours when the levels were very high. The initial rapid release of antibiotics both alone and in combination occurred within the first 24 hours. This high initial release was followed by a sustained elution that progressively became constant and gradual as shown by the extrapolation of the elution per day of both drugs (Figure 3). The levels of gentamicin alone (2.7 µg/day) and vancomycin alone (3.1 µg/day) obtained after 10 days of elution seem below the susceptibility limit of the most common pathogens involved in orthopedic surgical infections (Figure 3). The release of vancomycin alone during the study period was higher than that of gentamicin alone, corresponding to higher initial amount of vancomycin in the cylinders (20 mg in comparison to 10 mg of gentamicin). The release of vancomycin from PMMA cement was lower when combined with gentamicin in comparison to vancomycin alone at all times considered, as shown in Figure 3 and Table 1. FIGURE 3 - Elution (µg/day, extrapolation from Table 1) of gentamicin alone ( ), gentamicin in combination ( ), vancomycin alone ( ) and vancomycin in combination ( ) during 10 days, determined using FPIA method. The release of gentamicin and vancomycin in combination from PMMA cement showed a different pattern from that of the antibiotics alone. In the combination, we observed a decrease in the release of vancomycin (0.51%) from cement as compared to the release of vancomycin alone (1.16%), while the release of gentamicin was constant in both conditions TABLE 1 - Recovery (µg) of gentamicin alone, vancomycin alone and the gentamicin-vancomycin combination at different elution times determined using FPIA method. Values are expressed as mean (± SD); n = 4 test specimens for each assay in duplicate. Recovery (%) is expressed as the total amount of gentamicin alone, vancomycin alone and gentamicin-vancomycin combination released from test specimens after 240 h of elution compared to the initial amount. Time of Gentamicin Vancomycin Combination sampling (h) Alone (mg) Alone (mg) Gentamicin (mg) + Vancomycin (mg) (6.4) 62.4 (8.0) 60.4 (30.0) 26.9 (8.8) (1.2) 38.0 (5.4) 16.4 (4.4) 16.2 (3.4) (2.6) 24.3 (2.9) 7.8 (2.9) 11.3 (3.3) (2.6) 25.3 (2.6) 7.9 (3.2) 11.9 (3.8) (9.5) 39.6 (2.0) 19.6 (2.0) 20.8 (7.8) (3.6) 20.9 (4.5) 12.8 (3.0) 10.9 (4.9) (6.7) 21.5 (4.3) 15.6 (4.6) 4.1 (3.2) Cumulative elution (28.3) (14.1) (40.9) (39.4) Recovery (%) 1.55% 1.16% 1.41% 0.51%
6 RELEASE OF ANTIBIOTICS FROM POLYMETHYLMETHACRYLATE CEMENT 497 (1.55% gentamicin alone and 1.41% in the combination, respectively). The total release of the two antibiotics in combination (242.6 µg) was less than the expected theoretical release (388.5 µg) calculated as the sum of the concentrations of the single drugs. Therefore, the gentamicin+vancomycin mixture with PMMA seems to interfere negatively with vancomycin release from cement as shown by immunoenzymatic assay. Despite the impaired vancomycin release, the microbiological method showed that the antibiotics released from the cement were microbiologically active and exerted a synergistic effect on the microorganisms tested when in the combination. Simultaneously, the standard concentrations of the antibiotics utilized for microbiological assay were also determined by FPIA. Drug concentrations obtained by the FPIA method are overestimated, particularly at the lowest concentrations, as shown in Table 2. The overestimation of drug concentrations by FPIA should be caused by substances eluted from PMMA interfering with FPIA determination. According to the results obtained with FPIA for the release of gentamicin and vancomycin from PMMA we considered it more reliable to utilize the standard concentrations of the combination prepared in a ratio of 1:1. The initial ratio of 1:2 for gentamicin and vancomycin seems to overestimate the results of the elution. Therefore, the microbiological results reported here were obtained utilizing the standard curve of the 1:1 combination. Stability The solutions of gentamicin and gentamicinvancomycin combination were stable after 10 days at 37 C, while vancomycin showed a 15.4% loss of microbiological activity in 3 days and a 34.1% loss of activity after 10 days of incubation at 37 C (Figure 4a). The FPIA determination showed a loss of activity of vancomycin corresponding to 15.0% after 10 days of incubation. The antibiotics in combination presented slight modifications, within the range of normal fluctuations of the method (Figure 4b). Gentamicin and vancomycin, both alone and in combination, showed no changes in microbiological activity after 1 month at 4 C. DISCUSSION Antibiotic-impregnated PMMA cements are regarded as a safe method of delivering an antibiotic to the infection site, with a high initial release of drug followed by an elution that progressively diminishes over a period ranging from a few weeks to several months 8,9. Aminoglycosides, such as gentamicin, are TABLE 2 -Determination of standard concentrations of gentamicin alone, vancomycin alone and a gentamicin-vancomycin combination obtained with FPIA (mg/l). Values are expressed as mean (± SD). Concentrations tested below the limit of sensitivity are in boldface. ANTIBIOTIC ALONE COMBINATION Standard FPIA determination Standard FPIA determination concentration concentration (mg/l) Gentamicin + in PBS vancomycin (mg/l) Gentamicin Vancomycin Gentamicin Vancomycin (mg/l) (mg/l) (mg/l) (mg/l) (3.0) 40.7 (2.9) (0.1) 37.9 (5.3) (5.6) 22.4 (3.8) (2.0) 21.6 (2.8) (2.4) 11.9 (1.5) (3.0) 10.7 (1.9) (1.6) 6.4 (0.9) (0.9) 5.6 (0.8) (0.8) 3.6 (0.8) (0.7) 3.4 (0.4) (0.5) 2.7 (0.6) (0.4) 2.1 (0.4) (0.0) 1.9 (0.0) (0.4) 1.4 (0.2)
7 498 E. BERTAZZONI MINELLI - C. CAVEIARI - A. BENINI considered the most effective antibiotics to be used in PMMA cements because of their high solubility, their heat stability and their bactericidal activity at low concentrations. Moreover, aminoglycosides are effective against the majority of the most common microorganisms isolated from orthopedic infections, such as Staphylococcus aureus, Pseudomonas aeruginosa and enterococci 10. Vancomycin presents chemical characteristics similar to those of gentamicin, but is mainly considered the drug of choice in the presence of infections caused by methicillin-resistant staphylococci and S. epidermidis, which are bacteria frequently involved in orthopedic infections 3,11. Gentamicin and vancomycin in combination seem to be suitable antibiotic agents for use in PMMA bone cement by virtue of the following characteristics: stability of the drugs at body temperature, water-solubility to permit the diffusion of antibiotic from the cement, low incidence of hypersensitivity reactions and heat stability at temperatures up to 100 C (which frequently occur when the powdered polymer catalyst is mixed with the liquid monomer to form the bone cement) and bactericidal activity at low concentrations. Log concentration Log concentration 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 time 0 24 h 72 h 240 h A - Vancomycin alone ,8 Inhibition zone (mm) 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 time 0 24 h 72 h 240 h FIGURE 4- Antimicrobial activity after different periods of incubation at 37 C of A) vancomycin alone and B) vancomycin and gentamicin in combination expressed as inhibition zone (halo diameter) (B. subtilis microorganism-test). Vancomycin alone had reduced antimicrobial activity after 10 days of immersion. In our study we confirmed that gentamicin and vancomycin alone and in combination maintain their bioactivity after incorporation into PMMA cement. The antibiotic release is biphasic, showing good, prompt release from cement in the first 24 hours of elution with a gradual release over the following days. These results confirm the studies on elution kinetics of aminoglycosides from acrylic bone cements 9,12,13. Moreover, the antimicrobial activity of the combination shows a synergistic effect against microorganisms such as E. faecalis and E. coli. The effect of the combination is synergistic against enterococci 14 and variable against S. aureus according to strain 15, while the increased activity of gentamicin against E. coli in the presence of vancomycin is unexpected. However, we observed that the antimicrobial activity of the combination against E. coli was much higher than that of gentamicin alone (vancomycin alone was ineffective), presuming a synergistic effect evaluated in solid medium. These results were confirmed by checkerboard method, where the FIC index for E. coli (0.136) was below 0.5, meaning a synergism. The combination of gentamicin, inhibitor of protein synthesis, along with vancomycin, inhibitor of cell wall synthesis, could exert a positive effect. The presence of vancomycin, which is ineffective per se against Gram-negative bacilli, could induce different metabolic or morphological modifications in E. coli increasing the antimicrobial activity of gentamicin. Determination with FPIA and microbiological methods revealed different release rates of gentamicin and vancomycin from PMMA cement when the drugs are used alone or in combination. Vancomycin eluted less effectively than gentamicin from PMMA cement (Cemex ). These results are consistent with data obtained utilizing different acrylic cements (Palacos and Simplex) from other authors 12,13,16. The difference between elution capabilities may depend on more factors, such as physicochemical characteristics of vancomycin, molecular weight and interference with cement polymers, stability of drug to heat and to biological fluids as well as the different consistency of cement itself (degree of porosity, roughness, size of preparation and surface area, etc.). Results from the literature are difficult to compare because of different elution methods, type
8 RELEASE OF ANTIBIOTICS FROM POLYMETHYLMETHACRYLATE CEMENT 499 of cement and size of specimens 17,18. Vancomycin alone was released from PMMA cement in sufficient amount but its antimicrobial activity was low, exceeding the MIC 90 for E. faecalis in eluate for less than 24 hours. Vancomycin release from PMMA cylinders impregnated with the combination was lower (0.51%) than that from cylinders with vancomycin alone (1.16%), showing that combining vancomycin with gentamicin in PMMA cement induced certain modifications interfering with the release of vancomycin from the cement. Vancomycin did not affect the elution of gentamicin from cement. Gentamicin concentrations exceeded the E. coli MIC 90 for at least 96 hours; the combination exceeded the MIC 90 for E. faecalis for at least 72 h. The combination, however, maintains strong synergistic antimicrobial activity, counterbalancing the reduced release of vancomycin. Our results, according to Klekamp et al. 13, confirm the difficulty of eluting vancomycin; in contrast, we noted a further decrease in the amount of vancomycin eluted from cement in the presence of gentamicin. The elution of gentamicin and vancomycin from PMMA cement should improve by increasing the initial drug concentration in the cement. However, the amount of antibiotic should not modify the mechanical properties of cement. The results obtained with microbiological or FPIA method showed a constant overestimation of drug concentration when determined with FPIA method. The vancomycin concentration seems overestimated when compared to antimicrobial activity. We believe that it is possible that vancomycin breaks down to CDPI and CDPII during the mixture with PMMA and exothermic reaction. The elution time seems a minor factor of breakdown, mainly in the first phase of determination. The polymerization reaction and/or temperature should produce degradation products which are structurally similar to vancomycin, but not active microbiologically. Moreover, a temperature- and timedependent loss of activity of vancomycin in 240 h at 37 C was observed in our experimental conditions, confirming results obtained from other authors in different conditions when assayed with both the microbiological method and FPIA assay. The sterilization with ethylene dioxide and successive degasification (as for all items sterilized for human use with ethylene dioxide) should not interfere with the antimicrobial activity of both drugs. The microbiological determination is indicative of antimicrobial activity; the rate of inactivation of vancomycin alone and in combination is higher (0.77%) when determined with the FPIA method. These data and methods are in agreement with other data reported in the literature, showing that blood concentrations of vancomycin determined with the FPIA method were higher than those obtained with EMIT 22. The amount of vancomycin released at the infection site, however, is enough to exert good antimicrobial activity with a synergistic effect, when combined with gentamicin. Novel and recent local drug delivery systems are proposed to improve the local drug release i.e. biodegradable carriers for different drugs 12,17,23 with promising results, requiring further specific evaluation. In addition, local and systemic tolerability should be evaluated. The results obtained with test specimens are indicative of the preparation of cements or other biocompatible materials to be used in human subjects (for different types of prostheses). The microbiological method may be considered predictive of antimicrobial activity and efficacy of antibiotics released from PMMA cement at the infection site, as shown in preliminary clinical results 24. Gentamicin and vancomycin are both drugs responding to these requisites; gentamicin is present in a large number of commercial cements, confirming its validity. The increasing incidence of resistant Gram-positive bacteria requires the use of specific antimicrobial drugs. We stress that caution is necessary when mixing antibiotics in bone cement, especially when a combination of several drugs is made. It is always advisable to check for elution of drug from cement to be sure that the antibiotic-containing cement is effective and fulfills its aim. The hand mixture of antibiotics with bone cement by surgeons is an increasingly more frequent practice, which should be performed cautiously. These microbiological results seem to demonstrate the potential clinical efficacy of the vancomycin-gentamicin combination in treating local orthopedic infections caused by Grampositive methicillin-resistant microorganisms and coagulase-negative cocci.
9 500 E. BERTAZZONI MINELLI - C. CAVEIARI - A. BENINI ACKNOWLEDGEMENT: The authors wish to thank Sarah Turco for her excellent technical assistance. REFERENCES 1 Henry SL, Hood GA, Seligson D. Long-term implantation of gentamicin-polymethylmethacrylate antibiotic beads. Clin Orthopaed 1993; 295: Henry S, Galloway KP. Local antibacterial therapy for the management of orthopaedic infections. Pharmacokinetic considerations. Clin Pharmacokinet 1995; 29: Tunney MM., Ramage G, Patrick S, et al. Antimicrobial susceptibility of bacteria isolated from orthopaedic implants following revision hip surgery. Antimicrob Agents Chemother 1998; 42: Bassi C, Pederzoli P, Vesentini S, et al. Behavior of antibiotics during human necrotizing pancreatitis. Antimicrob Agents Chemother 1994; 38: National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically, 5 th ed. Approved standard. NCCLS document M7-A5. National Committee for Clinical Laboratory Standards, Wayne, PA Eliopolous GM, Moellering RC. Antimicrobial combination. In: Lorian V, ed. Antibiotics in Laboratory Medicine. 4th ed. Baltimore, Williams & Wilkins. 1996: Abbott Laboratories Diagnostic Division. TDx FLx system assay I. North Chicago, IL; Abbott Laboratories, Baystrom R, Milner RDG. The sustained release of antimicrobial drugs from bone cement. Br J Bone Joint Surg 1982; 5: Seyral P, Zannier A, Argenson JN. The release in vitro of vancomycin and tobramycin from acrylic bone cement. J Antimicrob Chemother 1994; 33: Sanderson PJ. Infection in orthopaedic implants. J Hospital Infect 1991; 18 (Suppl. A): Editorial. Born-again vancomycin. Lancet 1985; 1: Mader JT, Calhoun J, and Cobos J. In vitro evaluation of antibiotic diffusion from antibiotic-impregnated biodegradable beads and polymethylmethacrylate beads. Antimicrob Agents Chemother 1997; 41: Klekamp J, Dawson JM, Haas DW, DeBoer D, Christie M. The use of vancomycin and tobramycin in acrylic bone cement. Biochemical effects and elution kinetics for use in joint arthroplasty. J Arthroplast 1999; 14: Houlihan HH, Stokes DP, Rybak MJ. Pharmacodynamics of vancomycin and ampicillin alone and in combination with gentamicin once daily or thrice daily against Enterococcus faecalis in an in vitro infection model. J Antimicrob Chemother 2000; 46: Hershberger E, Aeschlimann JR, Moldovan T, Rybak MJ. Evaluation of bactericidal activities of LY333328, vancomycin, teicoplanin, ampicillin-sulbactam, trovafloxacin, and RP59500 alone or in combination with rifampin or gentamicin against different strains of vancomycin-intermediate Staphylococcus aureus by time-kill curve methods. Antimicrob Agents Chemother 1999; 43: Seligson D, Popham GJ, Voos K, Henry SL, Faghri M. Antibiotic-leaching from polymethylmethacrylate beads. J Bone Joint Surg 1993; 75: Kannellakopoulou K, Giamarellos-Bourboulis E. Carrier system for the local delivery of antibiotics in bone infections. Drugs 2000; 59: Wininger DA, Fass RJ. Antibiotic-impregnated cement and beads for orthopedic infections. Antimicrob Agents Chemother 1996; 40: Greenberg RN, Saeed AMK, Kennedy DJ, McMillan R. Instability of vancomycin in infusaid drug pump model 100. Antimicrob Agents Chemother 1987; 31: White LO, Edwards R, Holt HA, Lovering AM, Finch RG, Reeves DS. The in-vitro degradation at 37 of vancomycin in serum, CAPD fluid and phosphate-buffered saline. J Antimicrob Chemother 1988; 22: White LO, Holt HA, Reeves DS, MacGowan AP. Evaluation of Innofluor fluorescence polarization immunoassay kits for the determination of serum concentrations of gentamicin, tobramycin, amikacin and vancomycin. J Antimicrob Chemother 1997; 39: Follin SL, Mueller BA, Scott MK, Carfagna MA, Kraus MA. Falsely elevated serum vancomycin concentrations in hemodialysis patients. Am J Kidney Dis 1996; 27: Kannellakopoulou K, Kolia M, Anastassiadis A et al. Lactic acid polymers as biodegradable carriers of fluoroquinolones: an in vitro study. Antimicrob Agents Chemother 1999; 43: Bertazzoni Minelli E, Benini A, Biscaglia R, Magnan B, Bartolozzi P. Release of gentamicin and vancomycin alone and in combination from polymethylmethacrylate (PMMA) cement. J Antimicrob Chemother 1999; 44 (Suppl A):
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