In-vitro bactericidal activity of cefpirome and cefamandole in combination with glycopeptides against methicillin-resistant Staphylococcus aureus

M. Bergeret and J. Raymond*

Service Microbiologie, Hüpital Saint Vincent de Paul, 82, avenue Denfert Rochereau, 75674 Paris Cedex 14, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The bactericidal activity in vitro of cefpirome plus either vancomycin or teicoplanin was compared with that of a cefamandole–vancomycin combination against ten clinical isolates of homogeneous methicillin-resistant Staphylococcus aureus. Cefpirome (0.125 x MIC) combined with vancomycin (0.5–2x MIC) or teicoplanin (0.5–4x MIC) acted synergically against the ten isolates. Similar effects were observed with the cefamandole–vancomycin combination, except that for one isolate, higher cefamandole concentrations (0.25–1x MIC) were required.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections has increased. Most of the strains involved are multidrug-resistant, but are susceptible to glycopeptides, which are the principal treatment for MRSA. However, glycopeptide monotherapy is often not entirely effective because of the very low rate at which these antibiotics kill bacteria. The efficacy of adding a second antibacterial has therefore been investigated in vitro. 1 The efficacy of ß-lactam compounds alone against these organisms is unclear, but their combination with other compounds, like glycopeptides, is synergic in vitro. 2 Synergy between cefpirome and vancomycin against enterococci and MRSA has been shown by the chequerboard method. 3 We obtained similar results using the time–kill method and found that the combination was both bactericidal and synergic against methicillin-sensitive and -resistant strains of S. aureus and coagulase-negative staphylococci. 4

This study focused on MRSA, comparing the cefpirome–vancomycin treatment with cefamandole–vancomycin. Cefamandole was used because it is very active against S. aureus. 5 The activity of a teicoplanin–cefpirome combination was then compared with that of the vancomycin–cefpirome combination because synergy between ß-lactams and teicoplanin has also been reported. 1


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates

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 Mueller–Hinton 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 Mueller–Hinton broth (10 mL) containing antibiotic to give 4 x 10 6 cfu/mL. The same amount of inoculum was added to antibiotic-free Mueller–Hinton 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 time–kill 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.


    Results and discussion
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 Abstract
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 Materials and methods
 Results and discussion
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The MIC ranges for the ten isolates were 1–4 mg/L of vancomycin and 0.5–8 mg/L of teicoplanin. MICs of cefpirome were 16–128 mg/L. Except for one case, the MICs of cefamandole were lower than those of cefpirome (8–64 mg/L).

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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Table. Dynamics of killing by cefpirome plus vancomycin and cefamandole plus vancomycin of ten MRSA isolates. Decreases in surviving bacteria in log10 cfu/mL are given.
 
Similarly, cefpirome, at a concentration of 0.125 x MIC, acted synergically with teicoplanin (0.5 x MIC (one isolate), 1 x MIC (six isolates), 2 x MIC (two isolates) or 4 x MIC (one isolate)). Synergic killing was observed after 18 h (four isolates), 24 h (three isolates) and 48 h (three isolates) (data not shown).

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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Figure. Interactions of antimicrobials in vitro against MRSA isolate no. 8. (a) {square}, cefpirome alone, 0.125 x MIC; {triangleup}, vancomycin alone, 0.5 x MIC; •, cefpirome and vancomycin combined; (b) {square}, cefpirome alone, 0.125 x MIC; {triangleup}, teicoplanin alone, 0.5 x MIC; •, cefpirome and teicoplanin combined; (c) {square}, cefamandole alone, 0.5 x MIC; {triangleup}, vancomycin alone, 0.5 x MIC; •, cefamandole plus vancomycin.

 
For eight isolates, increasing the concentration of cefpirome (from 0.25 x MIC to 0.5 x MIC) resulted in bacteria being killed more rapidly (three isolates) or being killed at the same rate, but with lower concentrations of vancomycin (five isolates) (results not shown).

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 cefpirome–vancomycin and cefamandole–vancomycin 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 cefotaxime–fosfomycin with cefpirome–fosfomycin.

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.


    Acknowledgments
 
We would like to thank Hoechst Marion Roussel Pharmaceuticals for financial support.


    Notes
 
* Corresponding author. Tel:+33-1-40-48-82-42; Fax: +33-1-40-48-83-18; E-mail: j.raymond{at}svp.ap-hop-paris.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Debbia, E., Pesce, A. & Schito, G. C. (1986). In vitro interactions between teicoplanin and other antibiotics against enterococci and staphylococci. Journal of Hospital Infection 7, Suppl. A, 73–7.[ISI][Medline]

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, 804–9.[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, 25–30.[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, 1067–71.[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 time–kill studies amenable to routine laboratory use. Journal of Antimicrobial Chemotherapy37 , 842–4.[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, 488–92.

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, 23–28 June 1991. Futuramed, Munich. Abstract 256.

10 . 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, 47–51[Abstract/Free Full Text]

Received 19 May 1998; returned 13 July 1998; revised 3 August 1998; accepted 16 September 1998