In vitro activity of linezolid alone and in combination with gentamicin, vancomycin or rifampicin against methicillin-resistant Staphylococcus aureus by time–kill curve methods

Cédric Jacqueline1, Jocelyne Caillon1, Virginie Le Mabecque1, Anne-Françoise Miègeville1, Pierre-Yves Donnio2, Denis Bugnon1 and Gilles Potel1,*

1 Laboratoire d’Antibiologie (UPRES EA-1156), UER de Médecine, 1 rue Gaston Veil, 44035 Nantes Cedex 01; 2 Laboratoire de Bactériologie, Hôpital Pontchaillou, Rennes, France

Received 25 October 2002; returned 8 November 2002; revised 14 January 2003; accepted 20 January 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activity of the oxazolidinone linezolid was studied alone and in combination with three antibiotics acting on different cellular targets. Oxazolidinones are bacterial protein synthesis inhibitors that act at a very early stage by preventing the formation of the initiation complex. Combinations of linezolid with gentamicin, vancomycin or rifampicin were evaluated against four methicillin-resistant Staphylococcus aureus strains, using killing curves in conjunction with scanning electron microscopy. Time–kill curves were performed over 24 h using an inoculum of 5 x 106– 1 x 107 cfu/mL. Linezolid was studied at concentrations of 1 x, 4 x and 8 x MIC, with partner drugs at 8 x MIC. Addition of linezolid resulted in a decrease of antibacterial activity for gentamicin and vancomycin, and linezolid was antagonistic to the early bactericidal activity of gentamicin. Linezolid, in combination with rifampicin, showed an additive interaction for susceptible strains and inhibited rifampicin-resistant variants. Linezolid plus rifampicin appeared to be the most active combination against methicillin-resistant S. aureus strains in time–kill experiments.

Keywords: oxazolidinones, in vitro susceptibility study, drug combination interactions


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The large number of infections caused by Gram-positive bacteria that are resistant to many commonly used antibiotics constitutes a major challenge for clinicians and microbiologists. Staphylococcus aureus remains a major nosocomial pathogen in human infections, and the recent appearance of methicillin-resistant S. aureus (MRSA) strains with reduced susceptibility to glycopeptides1 has emphasized the need for new active drugs. Oxazolidinones are a novel class of synthetic antimicrobials with potent activity on Gram-positive pathogens such as enterococci (including vancomycin-resistant strains), Streptococcus pneumoniae, coagulase-negative staphylococci and S. aureus, including methicillin-resistant strains.2 Linezolid, the first drug issued from this class, is active against Gram-positive bacteria and displays non-bactericidal, time-dependent activity in vitro on staphylococci.3,4 Oxazolidinones are bacterial protein synthesis inhibitors and act at a very early stage by preventing the formation of the initiation complex.57 This mechanism of action is specific to this class, and no cross-resistance with other antimicrobial agents has been observed. Linezolid has been approved by US and European agencies and is indicated for the treatment of infections caused by Gram-positive pathogens in adult patients.8

Few reports have discussed the in vitro activity of combinations including linezolid.9,10 Studies are needed to assess the activity of combinations including linezolid against multiresistant Gram-positive bacteria. The present work investigated the in vitro activity of linezolid alone and in combination with gentamicin, vancomycin or rifampicin in order to determine the most active therapy against MRSA strains. In addition, scanning electron microscopy was performed to compare bacterial morphological alterations owing to the different combinations.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

The four MRSA strains studied were all isolated from blood cultures. One was a heterogeneous glycopeptide-intermediate strain (hGISA).

Antibiotics

Linezolid (Pharmacia Upjohn, Kalamazoo, MI, USA), vancomycin (Lilly, Saint Cloud, France), gentamicin (Schering Plough, Herouville Saint Clair, France), and rifampicin and teicoplanin (Aventis, Paris, France) were all provided by the manufacturers.

Medium

Mueller–Hinton (MH) broth (Sanofi Diagnostics Pasteur, Marne-la-Coquette, France) supplemented with calcium (25 mg/L) and magnesium (12.5 mg/L) was used for susceptibility tests and killing curve experiments. Colony counts were determined with MH plates (Difco) for time–kill experiments.

Susceptibility testing

MICs for the four strains were determined in cation-supplemented MH broth using the microdilution technique.11,12 Overnight MH broth cultures were used to prepare inocula of 105 cfu/mL. The MIC was defined as the lowest concentration of an antimicrobial agent that prevented turbidity when assessed after 24 h of incubation.

Time–kill curves

Killing experiments were performed to evaluate the antibacterial activity of combinations of antibiotics including linezolid against four MRSA strains. Linezolid was studied at 1 x, 4 x and 8 x MIC, in combination with partner drugs at concentrations of 8 x MIC. Time–kill curves were performed in glass flasks containing MH broth, using an inoculum of 5 x 106–1 x 107 cfu/mL in the presence of a single antibiotic or a combination of antibiotics.13 A flask of inoculated cation-adjusted MH broth with no antibiotic served as control. Tests were carried out in triplicate. Surviving bacteria were counted after 0, 3, 6 and 24 h of incubation at 37°C by subculturing 50 µL serial dilutions (10–1, 10–2 and 10–4 in order to eliminate potential carryover effect) of samples (in 0.9% sodium chloride) on MH plates using a spiral plater (Spiral System; Interscience, Saint-Nom-La-Bretèche, France). A bactericidal effect was defined as a >=3 log10 cfu/mL decrease compared with the initial inoculum after 24 h of incubation. Synergy was defined as a decrease of >=2 log10 cfu/mL between the combination and the most active single agent. Antagonism was defined as an increase in the colony count of >=2 log10 cfu/mL with the combination compared with the count obtained with the most active single agent.14

Scanning electron microscopy

Bacteria (overnight bacterial culture diluted to obtain 107 cfu/mL) were cultured for 24 h in MH broth containing linezolid alone (at 8 x MIC) or in combination with gentamicin, vancomycin or rifampicin (at 8 x MIC). Then, MH broth was centrifuged (3000g, 15 min, 4°C) before the pellets were mounted on membrane filters (Anodisc; Whatman International Ltd, Maidstone, UK). After 1 h, the filters were gently washed three times for 10 min in phosphate-buffered saline (PBS). Bacteria were fixed with 0.25% glutaraldehyde for 30 min before being washed several times in PBS. The cells were dehydrated through a graded ethanol series, and alcohol–freon substitution was performed. Before examination under a scanning electron microscope (JEOL 6400F), specimens were coated with 100 Å of a gold–palladium mix in an ion sputter (JEOL JFC 1100).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility tests

The MICs for S. aureus strains are summarized in Table 1. All strains were susceptible to linezolid. The hGISA strain showed increased MICs of vancomycin and teicoplanin. The other strains were susceptible to glycopeptides.


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Table 1.  MICs of study drugs for MRSA strains
 
Time–kill curves

The time–kill curves of linezolid plus gentamicin against the four strains are shown in Figure 1. Gentamicin alone was the most effective drug against the three gentamicin-susceptible strains, with bactericidal activity (>=3 log10 cfu/mL decrease) evident as early as 6 h. For the gentamicin-resistant hGISA strain, only a 1 log10 cfu/mL decrease was observed at 6 h, followed by regrowth. For all strains, linezolid, when added to gentamicin, was dominant,15 inhibiting the early bactericidal activity of gentamicin, particularly over the first 6 h. During this interval, inhibition of the bactericidal activity of gentamicin was dependent on the linezolid concentration. Although with decreased intensity, gentamicin killing occurred at 24 h. Thus, the addition of linezolid to gentamicin was antagonistic except with the hGISA strain, for which linezolid inhibited bacterial regrowth at 24 h.



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Figure 1. Antibacterial activity of linezolid (LNZ) alone and in combination with gentamicin (GEN) against (a) NA8, (b) BCB8, (c) P9 and (d) hGISA strains. Control (white circles); LNZ at 8 x MIC (black circles); GEN at 8 x MIC (white triangles); LNZ (1 x MIC) and GEN (8 x MIC) (black triangles); LNZ (4 x MIC) and GEN (8 x MIC) (white diamonds); LNZ (8 x MIC) and GEN (8 x MIC) (black diamonds). Error bars represent standard deviations.

 
Vancomycin, a time-dependent antibiotic, exhibited antibacterial activity (a 2.3–3.5 log10 cfu/mL decrease) within 24 h against NA8, BCB8 and P9 strains (Figure 2). As with gentamicin, addition of linezolid decreased the killing rate 100- to 1000-fold (except for the hGISA strain) compared with that obtained by vancomycin alone. Vancomycin activity was decreased on the hGISA strain (only a 1 log10 cfu/mL decrease in 24 h). Linezolid alone (at 8 x MIC) seemed to be more effective on the hGISA strain compared with the results obtained with linezolid alone on NA8, BCB8 and P9 strains. For these three strains, the decrease in the initial inoculum was <=1 log10 cfu/mL after 24 h, whereas it was close to 2 log10 on the hGISA strain.



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Figure 2. Antibacterial activity of linezolid (LNZ) alone and in combination with vancomycin (VAN) against (a) NA8, (b) BCB8, (c) P9 and (d) hGISA strains. Control (white circles); LNZ at 8 x MIC (black circles); VAN at 8 x MIC (white triangles); LNZ (1 x MIC) and VAN (8 x MIC) (black triangles); LNZ (4 x MIC) and VAN (8 x MIC) (white diamonds); LNZ (8 x MIC) and VAN (8 x MIC) (black diamonds). Error bars represent standard deviations.

 
Regrowth of rifampicin-resistant mutants occurred for all strains incubated with rifampicin alone at 8 x MIC (Figure 3). The addition of linezolid prevented the selection of resistant mutants at 24 h, and a slight synergy was observed at 24 h on the P9 strain.



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Figure 3. Antibacterial activity of linezolid (LNZ) alone and in combination with rifampicin (RIF) against (a) NA8, (b) BCB8, (c) P9 and (d) hGISA strains. Control (white circles); LNZ at 8 x MIC (black circles); RIF at 8 x MIC (white triangles); LNZ (1 x MIC) and RIF (8 x MIC) (black triangles); LNZ (4 x MIC) and RIF (8 x MIC) (white diamonds); LNZ (8 x MIC) and RIF (8 x MIC) (black diamonds). Error bars represent standard deviations.

 
Scanning electron microscopy

MRSA strains with linezolid alone or combined with gentamicin, vancomycin or rifampicin were photographed by electron microscopy to compare morphological alterations (Figure 4). For each combination, the most representative photograph was chosen, even if morphologically normal organisms were also observed. Linezolid alone (at 8 x MIC) exhibited moderate alterations on all strains studied. No cell destruction was observed, but only abnormal forms were visible (Figure 4b). However, gentamicin (at 8 x MIC) had a profound effect on the morphological structure of bacteria, and bacterial lysis was observed for most of the cells (Figure 4c). The combination of both agents showed abnormal forms without separation of the central septum, and no bacterial lysis was observed (Figure 4d).



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Figure 4. Scanning electron micrographs of BCB8 strain exposed to linezolid (LNZ) alone and in combination with gentamicin (GEN), vancomycin (VAN) or rifampicin (RIF), all at 8 x MIC. (a) Control, (b) LNZ, (c) GEN, (d) LNZ and GEN, (e) VAN, (f) LNZ and VAN, (g) RIF, (h) LNZ and RIF.

 
Vancomycin had an effect on the morphological structure of the cell, probably because of alteration of the cell wall, and inhibited cell division (Figure 4e). These effects did not seem to occur with the linezolid plus vancomycin combination (Figure 4f), and the bacterial cells observed were similar to those with linezolid alone.

For rifampicin alone or in combination with linezolid (Figure 4g and h), two types of cells were observed after 24 h of incubation: some showing only minor morphological alterations, and others that had undergone bacterial lysis. Thus, no differences were apparent between rifampicin alone and in combination with linezolid.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Linezolid, a new drug with potent activity on Gram-positive pathogens such as MRSA, displays non-bactericidal, time-dependent activity in vitro on staphylococci.2,4 In clinical settings, linezolid may be combined with other antimicrobial agents in order to increase the bactericidal activity of therapy, prevent the emergence of drug-resistant subpopulations and provide a complementary antibacterial spectrum.16 Zurenko et al.,2 in in vitro studies, found a low spontaneous mutation frequency (<8 x10–11) in S. aureus at 2 x, 4 x and 8 x MIC of linezolid, without rapid development of resistance. Clinical reports indicate that linezolid resistance is observed mainly in Enterococcus species.17 Only one report has concerned an S. aureus strain,18 and in all cases patients were treated for at least 3 weeks before linezolid resistance occurred. Thus, the study of combinations including linezolid would seem particularly important to ensure its future effectiveness.

In the present study, time–kill experiments were used to assess the activity of combinations. This technique, which measures bactericidal activity, appears to be more relevant than the chequerboard technique for clinical situations, in which bactericidal therapy is desirable.19 Moreover, killing curves provide a dynamic picture of antimicrobial action and interaction over time, as opposed to the chequerboard, which is usually applied only once (after 24 h of incubation).16 A starting inoculum of 5 x 106–1 x 107 cfu/mL was chosen as representative of those observed in severe clinical infections. The companion agents were tested at 8 x MIC in all cases, although gentamicin and rifampicin concentrations (for the hGISA strain) were not clinically achievable levels. However, rifampicin concentration was deliberately limited to 64 mg/L and these concentrations were tested in order to obtain a valid comparison between the different strains.

Linezolid and gentamicin are both ribosome-targeted compounds, but do not act at the same point in the ribosome cycle. Linezolid inhibits bacterial protein synthesis through a mechanism of action different from that of other antimicrobial agents. This drug binds to a site on the bacterial 23S ribosomal RNA of the 50S subunit and prevents the formation of a functional 70S initiation complex. In the same way, gentamicin disrupts normal protein synthesis by irreversible binding to the ribosome. In time–kill curves, the combination appears to be antagonistic, mainly for the early bactericidal activity of gentamicin. Linezolid is active upstream from other ribosome-targeted antibiotics (such as gentamicin) at a very early stage in the bacterial translation process. The formation of an initiation complex seems to be crucial for the bactericidal activity of aminoglycosides.20 By preventing the formation of a functional 70S initiation complex, linezolid could block the critical site of gentamicin action and inhibit its in vitro bactericidal activity. Another possibility is that linezolid inhibits the active transport mechanisms required for energy-dependent uptake of aminoglycosides in bacterial cells, as previously described for tetracycline and chloramphenicol.21 Despite antagonism being observed in the first 6 h, the combination seemed to suppress the emergence of gentamicin-resistant organisms, as observed on the hGISA strain (Figure 1d). This effect appeared to be similar to those observed when clindamycin is combined with aminoglycosides.22 Utilizing the time–kill curves technique, Zinner et al.23 have demonstrated antagonism (on the early killing effect) between amikacin or gentamicin with clindamycin, despite this combination having been shown to be synergic using the chequerboard technique against certain Enterobacteriaceae and Pseudomonas aeruginosa strains.24 However, the clinical significance of these observations remains to be established. These data support the need for in vivo investigations to validate the interaction observed in vitro between linezolid and aminoglycosides.

Vancomycin is a cell wall synthesis inhibitor, which means that bacteria must be in the growth phase to be subject to its bactericidal activity.25 Linezolid is a bacteriostatic agent, and its action on the ribosome inhibits bacterial growth. Consequently, the bactericidal activity of vancomycin could be partially inhibited by linezolid in a concentration-dependent manner such as that described above for gentamicin. Of particular interest was our finding of enhanced killing with linezolid on the hGISA strain. This enhanced effect has been reported on another GISA strain in an in vitro pharmacodynamic model.10 Nonetheless, further study would be beneficial in order to confirm these results on more GISA strains.

High-level resistant mutant selection limits the use of rifampicin as a single drug. Moreover, regrowth of rifampicin-resistant subpopulations always occurred in our study when this antibiotic was used alone. For all strains, linezolid combined with rifampicin inhibited the regrowth of rifampicin-resistant mutants. Moreover, an improvement of the antibacterial activity was observed with the combination compared with the most active single agent (i.e. linezolid). It was particularly important on the P9 strain with an increase close to 2 log10 cfu/mL compared with linezolid alone. Linezolid targets the formation of the initiation complex composed of 30S and 50S ribosome units, mRNA and N-formylmethionyl-tRNA. Rifampicin, by binding to DNA-directed RNA polymerase, prevents elongation of the RNA chain and stops bacterial growth.26 When the two drugs are combined, rifampicin normally acts before linezolid in the ribosome cycle and might prevent linezolid action. Consequently, the antibacterial activity observed in time–kill curves during the first 6 h was probably the result of rifampicin alone. Owing to the appearance of in vitro resistant variants, the antibacterial activity of the combination over the 6–24 h period is probably due to the action of linezolid alone. Linezolid takes over from rifampicin on RNA polymerase-muted bacteria by acting later in the ribosome cycle.

Jones et al.27 have investigated the activity of a new oxazolidinone, AZD2563, alone and in combination with gentamicin or vancomycin against staphylococci. Linezolid was used as a control. As in our study, no enhanced activity was observed with the addition of gentamicin or vancomycin to the oxazolidinones (AZD2563 and linezolid). No antagonism was noted in this study, probably because lower concentrations were used (0.25 x MIC versus 8 x MIC in our work). This study seemed to be well correlated with our observations.

Parallel qualitative electron microscopy experiments were performed to confirm the interactions observed with time–killing curves. The combination of gentamicin and linezolid appeared to be clearly antagonistic, with a lack of bacterial lysis compared with gentamicin alone. The effect on bacteria treated by linezolid plus vancomycin seemed to be similar to that with linezolid alone, which tends to confirm that linezolid partially inhibits the antibacterial activity of vancomycin (Figure 4e and f). No difference in morphological alteration was observed in electron microscopy when bacteria were treated by rifampicin alone or in combination with linezolid. Thus, electron microscopy observations seemed to be well correlated with the interactions observed in time–kill experiments.

In summary, linezolid plus rifampicin seemed to be the most active combination against MRSA strains in time–kill experiments. In clinical practice, antagonism is the most disadvantageous interaction possible with an antimicrobial combination. However, in vivo antagonism has rarely been documented in the literature,28 as compared with the large number of reports of in vitro antagonism. For example, rifampicin plus pefloxacin is usually antagonistic in vitro,29 but this interaction is not observed in vivo.30 Nevertheless, a recent study has reported that the combination of linezolid and vancomycin was less effective than vancomycin alone in an experimental endocarditis model due to MRSA.31 These data seem to confirm the interactions observed in our study. Consequently, in vivo studies are needed to validate the in vitro observations reported here with time–kill experiments. Finally, the good activity of linezolid on the hGISA strain suggests that this antibiotic would be a reasonable therapeutic choice for patients with limited possibilities of benefiting from other treatment.


    Acknowledgements
 
This work was supported by a grant from Pharmacia & Upjohn, Kalamazoo, MI, USA. This work was presented in part at the Forty-second Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 27–30 September 2002.


    Footnotes
 
* Corresponding author. Tel/Fax: +33-240-41-2854; E-mail: gpotel{at}sante.univ-nantes.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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