Department of Infectious Disease, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjyuku-ku, Tokyo 162-8666, Japan
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Abstract |
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Introduction |
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Infectious disease caused by MRSA often develops in compromised hosts with decreased neutrophil counts due to chemotherapy with antitumour drugs or decreased cellular immune function due to steroid hormone therapy, or in patients undergoing surgical procedures involving devices such as vascular catheters.
Although vancomycin, a glycopeptide antibacterial agent, shows good antibacterial activity against Gram-positive bacteria, and MRSA in particular, the effectiveness of vancomycin in these compromised hosts infected with MRSA is usually less than satisfactory.1 The rate of mortality from severe MRSA infection is reported to be as high as 1030% even after treatment with vancomycin.2 In MRSA infections, which include mixed infections with Gram-negative bacteria, the therapeutic effects of vancomycin alone are limited because it is not effective against Gram-negative bacteria. Combination therapy with other antibacterial agents should be attempted in such cases. Combination therapy may result in a lower dose of vancomycin and reduce adverse reactions such as nephrotoxicity.
The effects of combining vancomycin with other drugs have been studied by many investigators. Effective combinations of vancomycin with ß-lactams including carbapenem drugs have previously been reported.2,3,4 However, these results were generally obtained in vitro, and corresponding in-vivo effectiveness has not always been confirmed. Imipenem, a carbapenem, does not have bactericidal action against MRSA but it has a broad antibacterial spectrum covering both Gram-positive and Gram-negative bacteria, and has a stronger bactericidal activity than other ß-lactams. In the present study, we assessed the combined effects of imipenem/cilastatin plus vancomycin against MRSA infection in vivo using a neutropenic mouse model of thigh infection and against MRSA growth in vitro.
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Materials and methods |
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Vancomycin was obtained from Shionogi Seiyaku, Osaka, Japan. Imipenem and cilastatin were obtained from Banyu Seiyaku, Tokyo, Japan.
Determination of MIC
Bacteria cultured overnight on an agar plate and diluted with sterile physiological saline (107 cfu/mL) were used as the bacterial suspension. The suspension was added to cation-adjusted MuellerHinton broth (Nissui, Tokyo, Japan) containing the drug and incubated overnight at 35°C. MIC was determined by the microliquid dilution method.
Fractional inhibitory concentration index for the combined effects of drugs against MRSA strains isolated from clinical materials
Fractional inhibitory concentration (FIC) indices were obtained by the chequerboard method
for 36 MRSA strains isolated from clinical materials obtained at the hospital of Tokyo
Women's Medical College to assess the combined effects of vancomycin and imipenem.
Definitions based on this index were as follows: 0.5 was considered synergic; >0.5 to
1, additive; >1 to
2, independent and >2, antagonistic.5
Combined bactericidal effects in vitro
One strain (MRSA strain N), for which the MIC values of vancomycin and imipenem were 2 and 16 mg/L, respectively, was chosen from 36 clinical isolates. About 106 cfu/mL of MRSA strain N isolated from clinical materials were incubated in the presence of imipenem at 0.5 x MIC together with vancomycin at 0.25x, 0.5x, 1x, 4x or 16 x MIC for 0, 1, 2, 3, 4, 6 or 8 h. Aliquots of bacterial culture were plated on agar medium, and colonies were counted after incubation for 16 h at 37°C. Likewise, the bacterial growth was assessed in the presence of vancomycin 4 x MIC together with imipenem 0.2516 x MIC.
Postantibiotic effect in vitro
Postantibiotic effect (PAE) values were obtained by incubating MRSA strain N for 2 h in the presence of either vancomycin or imipenem at concentrations of 0.54 x MIC. Bacteria were washed three times on a membrane filter (pore size, 0.22 µm, Nippon Millipore, Japan), and transferred to fresh medium. During incubation, aliquots were removed from the culture at the times indicated and plated on agar medium, and colonies were counted after 24 h incubation. PAE values for combinations of the two drugs were obtained in the presence of vancomycin at 2 x MIC with imipenem at 14 x MIC or imipenem at 2 x MIC with vancomycin at 14 x MIC. PAE was defined as the time required for one unit of logarithmic growth in the presence of drug minus the time required for that growth in the absence of drug.
Bactericidal effects of drugs in the neutropenic mouse model of MRSA infection
To induce a decrease in the leucocyte count, cyclophosphamide at doses of 150 and 100 mg/kg was administered to 5-week-old female ICR mice 4 days and 1 day, respectively, before inoculation with MRSA. Exponentially growing MRSA suspended in 3% mucin were inoculated at a concentration of 5 x 105 cfu to both thighs of each animal. According to protocol 1, 2 h after inoculation, vancomycin at a dosage of 1 or 2 mg/kg and imipenem/cilastatin at a dosage of 5 or 10 mg/kg were injected sc in the back region hourly, either alone or in combination, for a total of eight injections. According to protocol 2, 2 h after inoculation, vancomycin at 4 mg/kg and imipenem/cilastatin at 20 mg/kg were injected simultaneously every 2 h for a total of four injections. According to protocol 3, 2 h after inoculation, the same doses of vancomycin and imipenem/cilastatin as in protocol 2 were injected individually every hour in sequence starting with vancomycin, for a total of eight injections. Protocol 4 was identical to protocol 3 except that imipenem/cilastatin was initially administered 2 h after MRSA inoculation. Time course assessments of MRSA proliferation in the thigh were estimated by collecting bacteria from homogenates of thigh tissue excised from animals at each time point as specified, and aliquots were plated on agar medium for colony assay. Suppressive time was defined as the duration for which bacteria were suppressed below the level just before therapy.
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Results |
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Synergic effects of the two drugs were observed against 34 of the 36 (94.4%) MRSA strains isolated from clinical materials. Additive effects were observed against the remaining two strains. The extent of the combined effect was more significant when imipenem was combined with various doses of vancomycin, i.e. MIC values of 0.3950 mg/L were obtained for imipenem alone in the medium, whereas values of 0.056.25 mg/L were obtained for the addition of vancomycin to imipenem (Table I). When the reverse sequence was used, the extent of the combined effect was not significant, i.e. MIC values of 0.391.56 mg/L were obtained for vancomycin alone, whereas values of 0.11.56 mg/L were obtained for the addition of imipenem to vancomycin (Table II).
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Using MRSA strain N, MIC values of 2 and 16 mg/L were obtained for vancomycin and imipenem, respectively. When about 106 cfu/mL of MRSA strain N in log phase were exposed to imipenem 0.5 x MIC together with vancomycin 0.25x, 0.5x, 1x, 4x or 16 x MIC, the combined effects of vancomycin did not vary in a MIC-dependent manner. However, bacterial regrowth observed in the presence of vancomycin 0.25x and 0.5 x MIC was no longer observed when imipenem 0.5 x MIC was added to vancomycin (Figure 1). When the bacteria were exposed to vancomycin 4 x MIC together with imipenem 0.25x, 0.5x, 1x, 4x or 16 x MIC, the bactericidal effect of imipenem was concentration dependent, and regrowth of bacteria which was observed in the presence of imipenem alone, was suppressed by the addition of vancomycin 4 x MIC, although a combined bactericidal effect with vancomycin 4 x MIC was not evident (Figure 2).
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PAE values obtained in vitro were 1.92.6 h for vancomycin and 2.63.5 h for imipenem (Table III). Values of 2.74.4 h were obtained for the combination of vancomycin and imipenem. The combined growth retardation effect tended to depend on the concentration of imipenem with a constant vancomycin concentration of 2 x MIC. In contrast, the combined effect of the reverse sequence with a constant imipenem concentration was not significant (Table IV).
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A single administration of vancomycin at 1 or 2 mg/kg or of imipenem/cilastatin at 5 mg/kg produced growth curves similar to those of the control, with a suppressive time of 0 h. After combined administration of these drugs, suppressive times were 2.9 h for 1 mg/kg of vancomycin plus 5 mg/kg of imipenem/cilastatin, 3.1 h for 10 mg/kg of imipenem/cilastatin alone, 3.9 h for 1 mg/kg of vancomycin plus 10 mg/kg of imipenem/cilastatin and 5.2 h for 2 mg/kg of vancomycin plus 10 mg/kg of imipenem/cilastatin (Figures 3 and 4).
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Discussion |
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Synergic effects of combining ß-lactams with vancomycin have been reported in studies investigating drugs for clinical use in treating MRSA. The combined effects of vancomycin and imipenem are better than those of vancomycin and cephems according to the FIC Index.2 However, these results were often obtained in vitro and may not be confirmed in vivo. For example, Sorrell et al. failed to demonstrate clinical effectiveness of combination therapy with vancomycin and amikacin, even though synergic effects were demonstrated for this combination in vitro.6 Therefore, results obtained in vitro should always be confirmed in vivo.
In the present study, in-vitro synergic effects of vancomycin and imipenem were observed for 34 of 36 (94.4%) MRSA strains isolated from clinical materials. Although the MICs of vancomycin were not significantly decreased in the presence of imipenem, the combined effect was more significant in lowering the MIC of imipenem. Similar results were obtained for bactericidal activity as judged from the survival curves. However, in this case, the combined effects may have been due not to killing of bacteria, but to suppression of bacterial regrowth, which was observed when vancomycin was used alone at relatively low concentrations. When the bacteria were exposed to a constant concentration of vancomycin together with increasing concentrations of imipenem, the bactericidal effect was dependent on the imipenem concentration. When imipenem at 16 x MIC was used together with 4 x MIC of vancomycin, surviving colonies of the MRSA strain used in this study decreased to below detectable levels after 6 h of incubation. However, when MIC values for imipenem against many other MRSA strains are taken into consideration, the use of imipenem at concentrations as high as 16 x MIC may be clinically impractical. We conclude from the in-vitro results that the co-administration of vancomycin and imipenem in vivo may play a critical role, when drug concentrations in the body decrease to the threshold for MRSA regrowth, or in poorly penetrable sites such as lungs and bones.
The search for potentially useful drugs that exert combined effects with vancomycin has mainly been carried out in vitro, and studies of in-vivo effectiveness have often not been reported. The present in-vivo study demonstrated that this combination of drugs retarded regrowth of MRSA as assessed by suppressive time. Maximum achievable blood concentrations of vancomycin and imipenem were estimated to be 1 mg/L and 10 mg/L, respectively, in the present animal study. Since these concentrations are not strong enough to exert bactericidal effects individually, the combined effect seemed to be synergic. In considering a rational in-vivo treatment for MRSA using these agents, the key issue may be that the circulating concentrations of the two drugs should be maintained above the MIC for as long as possible. Another important issue may be that the imipenem concentration can be increased, since the bactericidal and bacteriostatic actions of imipenem against bacterial regrowth were shown to be concentration dependent. Although the mechanism of this synergic effect is not yet understood, it should be noted that the two drugs inhibit different steps in the biosynthesis of bacterial cell walls, i.e. vancomycin has been proposed to attack a certain reaction step that is positioned upstream of the mucopeptide cross-linking step that is inhibited by ß-lactam antibiotics.2 In the present study, simultaneous administration of the two drugs was shown to be more effective than alternate dosing. With regard to other studies of alternate dosing, the combined effect of fosfomycin and flomoxef was most significant when fosfomycin was administered first.7
The synergic effects of vancomycin and imipenem were confirmed in both in-vitro and in-vivo studies. Clinically, there is a possibility that this combination will reduce both the vancomycin dose and adverse reactions, and be effective against mixed infections caused with bacteria such as Pseudomonas aeruginosa or other Gram-negative bacteria.
From those points of view, the combination of the glycopeptide vancomycin and imipenem may provide patients with clinical benefits, although at high cost.
The effectiveness of vancomycin under these conditions has not been previously reported. Hence, this application of vancomycin warrants further investigation to establish a rational combined therapy with vancomycin and imipenem for MRSA.
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Notes |
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References |
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2 . Chang, S. C., Hsieh, W. C., Luh, K. T. & Ho, S. W. (1989). Effects of antibiotic combinations on methicillin-resistant Staphylococcus aureus in vitro. Taiwan I Hsueh Hui Tsa Chih 88, 48892.[Medline]
3 . Erjavec, Z., de Vries-Hospers, H. G., van Kamp, H., van der Waaij, D., Halie, M. R. & Daenen, S. M. (1994). Comparison of imipenem versus cefuroxime plus tobramycin as empirical therapy for febrile granulocytopenic patients and efficacy of vancomycin and aztreonam in case of failure. Scandinavian Journal of Infectious Diseases 26, 58595.[ISI][Medline]
4 . Matsumoto, T., Kubo, S., Haraoka, M., Takahashi, K., Tanaka, M., Sakumoto, M. et al. (1993). Combination chemotherapy for infections due to methicillin resistant Staphylococcus aureus with combination therapy by cefuzonam and fosfomycin or minocycline in the urologic field. Clinical Therapeutics 15, 81928.[ISI][Medline]
5 . Krogstad, D. J. & Moellering, R. C. (1986). Antimicrobial combinations. In Antibiotics in Laboratory Medicine.2nd edn, (Lorian, V., Ed.), pp. 53795. Williams & Wilkins, Baltimore, MD.
6 . Sorrel, T. C., Packham, D. R., Shanker, S., Foldes, M. & Munro, R. (1982). Vancomycin therapy for methicillin resistant Staphylococcus aureus. Annals of Internal Medicine 97, 34450.[ISI][Medline]
7 . Hasegawa, H. (1991). Combination therapy of fosfomycin and flomoxef in methicillin-resistant Staphylococcus aureus infections: an in vitro experimental study on order and dose. Chemotherapy 39, 77181.
Received 8 January 1999; returned 30 March 1999; revised 14 May 1999; accepted 27 May 1999