In vitro synergy between cefepime and vancomycin against methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis

Alain Lozniewski*, Christine Lion, Francine Mory and Michèle Weber

Laboratoire de Bactériologie, Hôpital Central, 29 Avenue du Maréchal de Lattre de Tassigny, 54035 Nancy Cedex, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activity of cefepime combined with vancomycin was assessed by the chequerboard method against 35 clinical isolates of methicillin-susceptible (MSSA, n = 8) or -resistant (MRSA, n = 10) Staphylococcus aureus and methicillin-susceptible (MSSE, n = 9) or -resistant (MRSE, n = 8) Staphylococcus epidermidis and S. aureus ATCC 25923 (MSSA). The combination was synergic against 16 isolates and additive/indifferent against 20. For 10 of the clinical isolates (two MSSA, three MRSA, two MSSE, three MRSE) and the reference strain, the interaction of cefepime and vancomycin was also determined by the time–kill method. Except for one MRSA isolate, synergic killing was demonstrated with clinically achievable concentrations of vancomycin (0.5–1 mg/L) and cefepime (methicillin-susceptible isolates: 0.5–1 mg/L; methicillin-resistant isolates: 2–64 mg/L).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During the last decade, infections caused by methicillin-resistant staphylococci, particularly hospital-acquired infections, have increased substantially. Although vancomycin is commonly used to treat severe infections caused by methicillin-resistant staphylococci, its use as monotherapy in compromised hosts may be ineffective. Thus, combinations of this glycopeptide with other drugs such as ß-lactams may be useful for treating such infections. It has been shown previously that combinations of glycopeptides and ß-lactams may have synergic activity against Gram-positive cocci such as enterococci and staphylococci in vitro.1–3

Cefepime is an injectable cephalosporin with a broad spectrum of activity against many bacterial species, including staphylococci.4 Husson et al.5 noted that cefepime reduced the minimum killing time for vancomycin against methicillin-resistant staphylococci in vitro. However, this study was performed using only a single concentration of cefepime (32 mg/L). We examined in this study the in vitro activity of cefepime combined with vancomycin using a larger range of concentrations of both antibiotics against methicillin-susceptible or -resistant isolates of Staphylococcus aureus (MSSA or MRSA) and Staphylococcus epidermidis (MSSE or MRSE).


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

Eighteen isolates of S. aureus (eight MSSA and 10 MRSA) and 17 isolates of S. epidermidis (nine MSSE and eight MRSE), obtained from individual patients at our hospital, as well as S. aureus ATCC 25923 (MSSA) were used. The susceptibility of these isolates to methicillin has been determined previously by the oxacillin disc diffusion method at 30°C.6 All methicillin-resistant isolates were homogeneously resistant with the exception of two heterogeneously resistant MRSA. All isolates were stored in brain–heart infusion broth containing 15% (w/v) glycerol at –80°C until use.

Antibiotics

Cefepime and vancomycin were supplied by Bristol-Myers Squibb (Paris, France) and Lilly (Saint Cloud, France), respectively.

MIC and chequerboard titration assays

MICs of each antibiotic were determined for all isolates by agar dilution on Mueller–Hinton agar.7 S. aureus ATCC 25923 was included as a control. The MIC was defined as the lowest concentration of antibiotic that inhibited visible growth after 24 h incubation at 30°C (cefepime) or 37°C (vancomycin). MICs of vancomycin were interpreted as recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie.6

Chequerboard synergy testing was performed for all strains by agar dilution on Mueller–Hinton agar.8 Both antibiotics were tested at concentrations ranging (two-fold dilutions) from 0.06 to 64 x MIC. Plates were inoculated using a multipoint inoculator (5 x 104 cfu/spot) and incubated at 30°C (methicillin-resistant isolates) or 37°C (methicillin-susceptible isolates) for 24 h. The fractional inhibitory concentration (FIC) index was calculated by summing the FICs (MIC of drug A in combination with drug B/MIC of drug B alone) of vancomycin and cefepime. FIC indices were interpreted as synergic if the values were <=0.5, additive or indifferent if the values were >0.5–2 and antagonistic if the values were >2.

Time–kill assays

For 10 selected clinical isolates (two MSSA, two homogeneous and one heterogeneous MRSA, two MSSE and three MRSE) and S. aureus ATCC 25923, time–kill curves were constructed. Time–kill assays were performed in Mueller–Hinton broth inoculated with each isolate to a final concentration of 5 x 106 cfu/mL. Cefepime and vancomycin were tested alone, or in combination, at concentrations of 0.25, 0.5, 1 and 2 x MIC for 0, 2, 4, 6, 24 and 48 h incubation at 37°C (methicillin-susceptible staphylococci) or 30°C (methicillin-resistant staphylococci). Aliquots (0.1 mL) of bacterial culture were serially diluted and plated on to Mueller–Hinton agar; colonies were counted after incubation for 24 h at 30°C or 37°C. Drug carryover was prevented by dilution (>10–1). Tests were performed in duplicate; results are expressed as mean log10 cfu/mL. Bactericidal activity was defined as a reduction in viable bacteria of >=3 log10 cfu/mL. Synergy and additivity/indifference were defined respectively as a >=2 log10 decrease and a <2 log10 change in the viable count at 24 h for organisms treated with the combination compared with the viable count in the presence of the more active of the two antibiotics used alone.8


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MIC and chequerboard titration assays

All isolates were susceptible to vancomycin with MICs of 0.5–2 mg/L. MICs of cefepime ranged from 0.5 to 4 mg/L for MSSE, from 1 to 4 mg/L for MSSA, and from 4 to 128 mg/L for both MRSE and MRSA. Against 16 clinical isolates (two MSSA, seven MSSE, three MRSA and four MRSE) a synergic bacteriostatic effect (FIC index range: 0.2–0.5) between cefepime and vancomycin was observed. For all the other isolates tested, an additive/indifferent effect (FIC index range: 0.6–1) was noted. Synergy was observed more frequently against MSSE (seven of nine isolates) than against MRSE (four of eight isolates). For S. aureus isolates, the synergy rate was not influenced by methicillin resistance.

Time–kill assays

At any concentration tested, no killing effect was observed at 2 and 4 h for any isolate. When methicillin-susceptible isolates were exposed to cefepime alone, a bactericidal effect was noted for all isolates with concentrations of 2 x MIC (1–4 mg/L) at 24 h except for one MSSA isolate (MSSA no. 1) for which no bactericidal effect was noted at this concentration. For methicillin-resistant isolates such activity was not observed. Vancomycin used alone was bactericidal for all isolates after 24–48 h of incubation at concentrations of 1–2 x MICs (1–4 mg/L) (Table IGo). Addition of cefepime at concentrations of 0.5–1 mg/L for all methicillin-susceptible isolates and of 4–128 mg/L for all but one of the methicillin-resistant isolates was bactericidal at 24 h with lower concentrations of vancomycin (0.5–1 mg/L) than those required to achieve such effect with vancomycin alone (1–4 mg/L). This synergy was also demonstrated between vancomycin (0.25–1 mg/L) and cefepime at concentrations <=1 mg/L for all methicillin-susceptible strains and at concentrations of 2–64 mg/L for five of the six methicillin-resistant strains tested (Table IIGo). At the lowest concentrations of vancomycin/cefepime yielding synergy, a reduction in viable count of >=3 log10 cfu/mL was always observed with the exception of three isolates (MSSA no. 3, MRSA nos 4 and 5) for which only a 2–2.5 log10 cfu/mL decrease was noted at 24 h. Against two MRSE isolates (MRSE nos 10 and 11) and one MSSA isolate (MSSA no. 2), the combination of cefepime at 2 x MIC (4–128 mg/L) with vancomycin at 1 or 2 x MIC (0.5–1 mg/L) achieved a bactericidal effect at 6 h instead of 24 h (Table IGo).


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Table I. Killing effect of vancomycin combined with cefepime on three isolates of MSSA (including S. aureus ATCC 25923), three MRSA, two MSSE and three MRSE
 

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Table II. Bacteriostatic and bactericidal effect of vancomycin combined with cefepime on methicillin-susceptible or -resistant S. aureus and S. epidermidis isolates
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
By the chequerboard method, synergy between cefepime and vancomycin was observed for nine (50%) of the 18 methicillin-susceptible staphylococci and for seven (38.8%) of the 18 methicillin-resistant staphylococci. Bactericidal synergy was demonstrated against 10 of the 11 isolates tested by time–kill assays. However, for four of these strains (two MSSA, one MRSA and one MRSE) the chequerboard method failed to detect synergy. Discrepancies between chequerboard and time–kill methods in the detection of synergy have been reported for several bacterial species including staphylococci.3,9 This may be explained, in part, by the fact that the chequerboard titration method is a qualitative test of bacteriostatic activity at only a single point in time, while the time–kill assay is a quantitative test of both bacteriostatic and bactericidal activities at different points in time. Moreover, definitions of synergy differ between these two methods. Nevertheless, it has been shown that synergy is best detected by the time–kill method, the results of which generally correlate better with improved clinical outcome than those of chequerboard titration results.9

We showed that the combination of cefepime with vancomycin allowed a reduction in the concentrations of vancomycin required to achieve bactericidal activity and induced bactericidal synergy for all methicillin-susceptible and for five of the six methicillin-resistant isolates tested. These results are similar to those obtained by Raymond et al.,2 who reported that cefpirome (4–32 mg/L), another broad-spectrum cephalosporin, acted synergically with vancomycin (2–4 mg/L) against all four MRSA isolates and all three methicillin-resistant coagulase-negative staphylococci tested. Husson et al.5 reported that when three MRSE isolates were exposed to cefepime at 32 mg/L combined with vancomycin at 1–2 mg/L, a bactericidal effect was obtained at 6 h (n = 2) or 24 h (n = 1) whereas vancomycin was bactericidal for only one strain at 48 h. We also found that for one MSSA and two MRSE isolates, the addition of cefepime yielded more rapid killing. The concentrations that potentiate the killing activity of vancomycin can be achieved in plasma, since after intravenous infusion of a 2 g dose, cefepime achieves high peak plasma concentrations of 126–193 mg/L and mean plasma concentrations of approximately 118, 72, 48 and 16 mg/L at 1, 1.5, 2.5 and 5.5 h, respectively.4,10

Considering the broad-spectrum antibacterial activity of cefepime and the potential enhancement of the antistaphylococcal activity of vancomycin by this ß-lactam antibiotic, the combination of these two antimicrobial agents might be useful in the treatment of severe staphylococcal infections as well as in the empirical treatment of mixed infections. However, larger in vitro and animal studies will be necessary to confirm these results.


    Notes
 
* Corresponding author. Tel: +33-3-83852196; Fax: +33-3-83852673; E-mail: a.lozniewski{at}chu-nancy.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Ravizzola, G., Cabibbo, E., Peroni, L., Longo, M., Pollara, P. C., Corulli, M. et al. (1997). In-vitro study of the synergy between ß-lactam antibiotics and glycopeptides against enterococci. Journal of Antimicrobial Chemotherapy 39, 461–70.[Abstract]

2 . 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]

3 . Barr, J. G., Smyth, E. T. M. & Hogg, G. M. (1990). In vitro antimicrobial activity of imipenem in combination with vancomycin or teicoplanin against Staphylococcus aureus and Staphylococcus epidermidis. European Journal of Clinical Microbiology and Infectious Diseases 9, 804–9.[ISI][Medline]

4 . Barradel, L. B. & Bryson, H. M. (1994). Cefepime. A review of its antibacterial activity, pharmacokinetic properties and therapeutic use. Drugs 47, 471–505.[ISI][Medline]

5 . Husson, M. O., Fruchart, A. & Izard, D. (1998). Etude comparative de l'activité bactéricide du céfépime et du cefpirome en association avec les glycopeptides vis-à-vis de staphylocoques sensibles et résistants à la méthicilline. Pathologie Biologie 46, 279–83.[ISI][Medline]

6 . Comité de l'Antibiogramme de la Société Française de Microbiologie. (1999). Communiqué 1999. Pathologie Biologie 47, 845–72.[ISI][Medline]

7 . Comité de l'Antibiogramme de la Société Française de Microbiologie. (1993). Recommandations du Comité de l'Antibiogramme de la Société Française de Microbiologie. Bulletin de La Société Française de Microbiologie 8, 156–66.

8 . Eliopoulos, G. M. & Moellering, R. C. (1996). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed.), pp. 330–96. Williams & Wilkins, Baltimore, MD.

9 . Johnson, C. C. (1996). In vitro testing: correlations of bacterial susceptibility, body fluid levels, and effectiveness of antibacterial therapy. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed.), pp. 813–34. Williams & Wilkins, Baltimore, MD.

10 . Nye, K. J., Shi, Y. G., Andrews, J. M. & Wise, R. (1989). Pharmacokinetics and tissue penetration of cefepime. Journal of Antimicrobial Chemotherapy 24, 23–8.[Abstract]

Received 23 June 2000; returned 7 August 2000; revised 30 August 2000; accepted 21 September 2000