Service Microbiologie, Hüpital Saint Vincent de Paul, 82, avenue Denfert Rochereau, 75674 Paris Cedex 14, France
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Abstract |
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Introduction |
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This study focused on MRSA, comparing the cefpiromevancomycin treatment with cefamandolevancomycin. Cefamandole was used because it is very active against S. aureus. 5 The activity of a teicoplanincefpirome combination was then compared with that of the vancomycincefpirome combination because synergy between ß-lactams and teicoplanin has also been reported. 1
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Materials and methods |
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Ten clinical isolates of S. aureus from three hospitals were included in this study. All isolates were uniformly resistant to methicillin in pure culture as shown by disc sensitivity testing at 30°C and 37°C.
Antibiotics
Cefpirome and teicoplanin were supplied by Hoechst Marion Roussel (Romainville, France), and cefamandole and vancomycin by Eli Lilly and Co. (Indianapolis, IN, USA).
Susceptibility testing
The MICs of cefpirome, cefamandole, vancomycin and teicoplanin were determined by a macrodilution procedure in MuellerHinton broth (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) using an inoculum of 4 x 106 cfu/mL. The MIC was the lowest concentration of the antibiotic that inhibited visible growth after 24 h incubation at 37°C.
Killing curves
Killing curve assays were performed with cefpirome and cefamandole, alone or in combination at concentrations of 0.062, 0.125, 0.5, 1 and 2 x MIC for each isolate. A mid-logarithmic phase culture was diluted in MuellerHinton broth (10 mL) containing antibiotic to give 4 x 10 6 cfu/mL. The same amount of inoculum was added to antibiotic-free MuellerHinton broth as a growth control. Aliquots (200 µL) from each tube were transferred to the wells of sterile microplates and the micro-method of Vedel et al. 6 was carried out. Aliquots were collected after 6, 18, 24 and 48 h and were transferred to blood agar. Plates were incubated for 18 h at 37°C and the percentage of surviving bacterial cells was determined for each sampling time by comparing colony counts with those for standard dilutions (10 1 to 10 5) of the initial bacterial inoculum. The results were plotted on semilog graph paper to obtain killing curves. Results are expressed as log10; cfu/mL of surviving bacterial cells. The growth controls became turbid after 24 h. All timekill tests were carried out in duplicate. Bactericidal synergy in vitro was defined as counts at least 2 log10; cfu/mL lower after 24 or 48 h treatment with the drug combination than with the most active single agent. A bactericidal effect was defined as a 3 log10; decrease in cfu/mL (99.9% kill). 7 Antibiotic carryover was not a problem in our determinations because it only occurs at higher concentrations (>16 x MIC). 7 We tested directly for drug carryover, and no effects were detected with concentrations used in this study.
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Results and discussion |
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Cefpirome acted synergically for the ten isolates at a concentration of 0.125 x MIC when combined with 0.5 x MIC (one isolate), 1 x MIC (seven isolates) or 2 x MIC (two isolates) of vancomycin. Synergic killing was observed after 18 h (two isolates), 24 h (five isolates) and 48 h (three isolates) (Table).
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In one case (isolate 6), synergic killing occured after 48 h with a cefamandole concentration of 0.125 x MIC combined with a vancomycin concentration of 2 x MIC. With vancomycin concentrations of 0.5 1 x MIC, the cefamandole concentration required for synergic killing was 0.25 x MIC for two isolates, and 0.5 or 1 x MIC for seven isolates. Synergic killing occured after 18 h (three isolates), 24 (two isolates) and 48 h (four isolates).
The bactericidal effects of the various combinations on isolate 8 are shown in Figure 1.
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MRSA infections are a major infectious disease problem. Treatment sometimes fails with vancomycin alone, especially in MRSA infections, so the optimum strategy is vancomycin combined with other antistaphylococcal agents. 8 Synergy between ß-lactams, such as imipenem or meropenem, and glycopeptides has been reported. 2 Synergy between cefpirome and vancomycin against MRSA was first reported by Seibert et al. 3 using the chequerboard procedure. These drugs are cell wall-active agents, so better results are obtained with exponentially growing cells rather than with stationary-phase staphylococcal inocula. Our results confirm that cefpirome acted in synergy against MRSA not only with vancomycin, but also with teicoplanin. Synergy between teicoplanin and ß-lactams was previously reported by Debbia et al.1 and Barr et al. 2 against methicillin-susceptible or -resistant S. aureus.
The cefpiromevancomycin and cefamandolevancomycin combinations had similar bactericidal effects, but higher concentrations (two to eight times) of cefamandole than cefpirome were required. The greater synergy against S. aureus of combinations with cefpirome was described by Drugeon et al. 9 who compared cefotaximefosfomycin with cefpiromefosfomycin.
Cefpirome and vancomycin killed staphylococci more rapidly than vancomycin alone. Cefpirome increased the activity of vancomycin by increasing both the rate and number of bacteria killed and prevented later regrowth.
Synergy between ß-lactams and glycopeptides against Gram-positive cocci has occasionally been reported, but the mechanism of synergy is unknown. A recent study by Sieradzki & Tomasz 10 showed that inhibitors of early steps in peptidoglycan synthesis, such as fosfomycin, D-cycloserine, vancomycin and teicoplanin greatly reduce methicillin resistance. Further studies must be undertaken to determine the mechanism of resistance which occurred at particularly low cefpirome concentrations.
For the ten isolates studied, higher concentrations of vancomycin and cefpirome can be achieved in the serum than were used in this study. This may be essential in deep-seated infections in which drug penetration is low. The combination of cefpirome with a glycopeptide may be of particular value in cases of severe or suspected mixed infections. Animal experiments are required to determine the treatment potential of these combinations.
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Acknowledgments |
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Notes |
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References |
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2 . Barr, J. G., Smyth, E. T. M. & Hogg, G. M. (1990). In vitro antimicrobial activity of imipenem in combination with vancomycin and teicoplanin against Staphylococcus aureus and Staphylococcus epidermidis. European Journal of Clinical Microbiology and Infectious Diseases 9, 8049.[ISI][Medline]
3 . Seibert, G., Isert, D., Klesel, N., Limbert, M., Markus, A. & Schrinner, E. (1992). The in-vitro antibacterial activity of a combination of cefpirome or cefoperazone with vancomycin against enterococci and Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 29, Suppl. A, 2530.[ISI][Medline]
4 . Raymond, J., Vedel, G. & Bergeret, M. (1996). In vitro bactericidal activity of cefpirome in combination with vancomycin against Staphylococcus aureus and coagulase-negative staphylococci. Journal of Antimicrobial Chemotherapy 38, 106771.[Abstract]
5 . Stratton, C. W., Liu, C. & Weeks, L. S. (1987). Activity of LY146032 compared with that of methicillin, cefazolin, cefamandole, cefuroxime, ciprofloxacin and vancomycin against staphylococci as determined by kill-kinetic studies. Antimicrobial Agents and Chemotherapy 31,1210 5.[ISI][Medline]
6 . Vedel, G., Bouchet, E., Gangneux, J. P. & Nevot, P.(1996). A simple micro-method for timekill studies amenable to routine laboratory use. Journal of Antimicrobial Chemotherapy37 , 8424.[ISI][Medline]
7 . National Committee for Clinical Laboratory Standards. (1992). Methods for Determining Bactericidal Activity of Antimicrobial Agents: Tentative Guideline M26-T. NCCLS, Villanova, PA.
8 . 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 [Journal of the Formosan Medical Association] 88, 48892.
9 . Drugeon, H. B., Caillon, J., Juvin, M. E. & Picault, J. L. (1991). Bactericidal activity of cefpirome-fosfomycin in comparison with cefotaxime fosfomycin against Staphylococcus aureus. In Abstracts of the Seventeenth International Congress of Chemotherapy, Berlin, 2328 June 1991. Futuramed, Munich. Abstract 256.
10
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Sieradzki, K. & Tomasz, A. (1997).
Suppression of
ß-lactam antibiotic resistance in a methicillin-resistant Staphylococcus aureus
through synergic action of early cell wall inhibitors and some other antibiotics. Journal
of Antimicrobial Chemotherapy 39, Suppl. A, 4751
Received 19 May 1998; returned 13 July 1998; revised 3 August 1998; accepted 16 September 1998