ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1992, p. 2611-2616
Vol. 36, No. 12
0066-4804/92/122611-06$02.00/0 Copyright © 1992, American Society for Microbiology
Daptomycin or Teicoplanin in Combination with Gentamicin for Treatment of Experimental Endocarditis Due to a Highly Glycopeptide-Resistant Isolate of Enterococcus faecium FRANCOIS CARON,"* MARIE-DOMINIQUE KITZIS,2 LAURENT GUTMANN,2 ANNE-CLAUDE CREMIEUX,3 BERNARD MAZIERE,4 JEAN-MARIE VALLOIS,3 AZZAM SALEH-MGHIR,3 JEAN-FRAN§OIS LEMELAND,1 AND CLAUDE CARBON3 Groupe de Recherche en Infections Experimentales, H6pital Charles Nicolle, 76031 Rouen Cedex, 1 Laboratoire de Microbiologie Medicale, Universite Pierre et Marie Curie, 75270 Paris Cedex 06,2 Institut National de la Sante et de la Recherche Medicale, Unite 13, Hopital Bichat-Claude Bernard, 75944 Paris Cedex 19,3 and Service Hospitalier Frederic Joliot, Orsay,4 France Received 11 May 1992/Accepted 31 August 1992
Using an experimental endocarditis model, we studied the activity of daptomycin used alone or in combination with gentamicin against an Enterococcusfaecium strain that was highly resistant to glycopeptides and susceptible to gentamicin. In vitro, the MIC of daptomycin was 1 ,ug/ml. In vivo, daptomycin appeared to be effective only when it was used in a high-dose regimen, i.e., 12 mg/kg of body weight every 8 h (-2.5 log1o CFU/g versus controls; P < 0.05), particularly when it was combined with gentamicin (-5.0 log1o CFU/g versus controls; P < 0.01). Since the distribution of daptomycin into cardiac vegetations, as evaluated by autoradiography, appeared to be homogeneous, the poor in vivo activity of daptomycin was considered to be related to its high degree of protein binding, as suggested by killing curves studies. Since the MIC of teicoplanin for the vancomycin-resistant E.faecium strain used in the study was only 64 ,ug/ml and since an in vitro synergy between teicoplanin at high dose and gentamicin was observed, a high-dose regimen of teicoplanin, i.e., 40 mg/kg every 12 h, was also assessed in vivo. This treatment provided marginal activity only when it was combined with gentamicin (-2.3 log1o CFU/g versus controls; P < 0.05). These results suggest that the levels of daptomycin or teicoplanin in serum required to cure experimental endocarditis caused by a highly glycopeptide-resistant strain of E. faecium would not be achievable in humans.
Glycopeptide-resistant strains of enterococci, mainly Enterococcus faecium, have been observed with increasing frequency (4, 11). Most of the strains express high-level resistance to vancomycin (MICs, 64 to > 1,000 ,ug/ml) and, to a lesser extent, to teicoplanin (MICs, 32 to 256 ,ug/ml). The synergistic effect that vancomycin and aminoglycoside combinations usually display against enterococci in vitro is constantly abolished against vancomycin-resistant strains (13, 18). In the absence of cross-resistance between glycopeptide and lipopeptide antibiotics, daptomycin remains effective in vitro against such strains (4). Treatment of infections caused by highly glycopeptideresistant enterococci is not well defined. We have recently demonstrated in vitro and in an experimental endocarditis model the potential efficacy of the triple combination of penicillin-vancomycin-gentamicin for the treatment of serious infections caused by moderately penicillin- and highly glycopeptide-resistant enterococci (3). However, such a treatment could not be given to patients allergic to ,B-lactam antibiotics; in that case, other treatments are warranted. The first aim of the study described here was to evaluate the efficacy of daptomycin used alone or in combination with gentamicin for the treatment of experimental endocarditis caused by the clinical isolate of Enterococcusfaecium D400, which is highly resistant to glycopeptides and susceptible to gentamicin (3). The results obtained in animals were analyzed according to the in vitro activity of the drug, its pharmacokinetic parameters determined in animals, and a *
quantitative autoradiographic analysis evaluating its diffusion into cardiac vegetations. Since the MIC of teicoplanin for E. faecium D400 was only 64 ,ug/ml and since a synergy between teicoplanin at high concentration and gentamicin was observed in vitro, a second aim was to evaluate in vivo the efficacy of a highdose regimen of teicoplanin (HiD teicoplanin) and to compare it with the efficacy of a lower-dose regimen (LoD teicoplanin) which provides levels in animal sera comparable to those achieved in humans by the currently recommended regimen. MATERIALS AND METHODS Bacterial strain. E. faecium D400 (1, 3) was isolated from the stool of an immunocompromised patient who underwent digestive decontamination with vancomycin. Antibiotics and media. Daptomycin, teicoplanin, and gentamicin were kindly supplied, respectively, by Eli Lilly (Saint-Cloud, France), Marion Merrell Dow (LevalloisPerret, France), and Schering Plough (Levallois-Perret, France). In vitro studies were performed by using Mueller-Hinton broth (Diagnostics Pasteur, Marne-la-Coquette, France) supplemented with calcium (50 jig/ml) and magnesium (25 ,ug/ml) for daptomycin and by using brain heart infusion broth (Difco, Detroit, Mich.) for teicoplanin and gentamicin. Susceptibility tests. MICs and MBCs were determined by the macrodilution method (16). Each value was determined with a final log-phase inoculum of 5 x 105 CFU/ml. The MIC was defined as the lowest concentration of drug that inhib-
Corresponding author. 2611
2612
CARON ET AL.
ited growth, i.e., visible turbidity after 24 h of incubation at 37°C. The MBC was determined by subculturing 10 p,l from each clear tube on agar plates and was defined as the lowest concentration that reduced the number of viable organisms by at least 99.9%. Killing curves. Killing curves were determined for daptomycin, teicoplanin, and gentamicin, which were used alone or in different combinations. Each value was determined with bacteria in the exponential growth phase by using a final inoculum of 5 x 107 CFU/ml in order to approximate in vitro the high bacterial concentrations observed in cardiac vegetations. The antibiotic concentrations were also chosen to reflect the levels and the ratios achieved with the various regimens (see below) in rabbit serum. These concentrations were 10 and 40 ,ug/ml for daptomycin, 20 and 90 ,ug/ml for teicoplanin, and 4 p,g/ml for gentamicin. Killing curves were also determined in the presence of 50% rabbit serum at the same final concentrations of each drug given above. Samples were removed at 0, 3, 6, and 24 h in order to determine, by serial dilutions and plating in duplicate, the number of viable bacteria. Synergy was defined as a decrease in the CFU per milliliter by at least 2 log1o units compared with the effect of the most active single component in the drug combination. Experimental endocarditis. A bacterial aortic endocarditis was established by the technique of Perlman and Freedman (15), as follows. On day 1, female New Zealand White rabbits were anesthetized by intramuscular injections with ketamine hydrochloride (15 mg/kg of body weight). A carotid artery was exposed and cannulated with a polyethylene catheter. The catheter was advanced until pulsations and resistance indicated that it had reached the apex of the left ventricle. It was then sealed with a ligature and was left in place for the duration of the experiment. Twenty-four hours after surgery, animals were injected in the marginal vein with a log-phase inoculum of E. faecium D400 of approximately 108 CFU in a volume of 1 ml. A total of six rabbits receiving no treatment were used as controls. Eighty-four animals were treated from days 4 to 8, i.e., for 5 days, and were assigned to one of the following nine groups: (i) LoD daptomycin, i.e., 10 mg/kg twice daily (BID); (ii) HiD daptomycin, i.e., 12 mg/kg three times daily (TID); (iii) LoD teicoplanin, i.e., 10 mg/kg BID; (iv) HiD teicoplanin, i.e., 40 mg/kg BID; (v) gentamicin, 6 mg/kg BID; (vi) LoD daptomycin plus gentamicin; (vii) HiD daptomycin plus gentamicin; (viii) LoD teicoplanin plus gentamicin; and (ix) HiD teicoplanin plus gentamicin. The first dose of daptomycin or teicoplanin was doubled for loading. On day 9, animals were killed when each drug concentration reached a trough, i.e., 8 h after the last injection of the antibiotic given TID (i.e., for HiD daptomycin) or 12 h after the last injection of the antibiotics given BID (i.e., for the other drug regimens), and the heart was removed and inspected. Some rabbits were excluded because of perioperative death, negative pretreatment blood cultures, early death during the -course of treatment, or incorrect placement of the catheter across the aortic valve. All vegetations from each animal were excised, pooled, weighed, and rinsed in sterile saline. Vegetations from each animal were homogenized in 0.5 ml of sterile saline, and 0.1-ml aliquots were quantitatively subcultured on brain heart infusion agar plates at 37°C for 24 h. Portions of each vegetation homogenate were also cultured by the disk diffusion method to confirm the persistence of the initial pattern of antibiotic susceptibility of E. faecium D400 after the therapy period. The lower limit of detection by this method was 100 CFU/g. This value of 2 log1o CFU/g was assigned to all sterile vegetations and was used for the
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. In vitro antimicrobial susceptibility of E. faecium D400 determined in the absence or presence of rabbit serum Antibiotic
Vancomycin Teicoplanin Daptomycin Gentamicin
Medium without serum
Medium with 50% serum
MIC (,ug/ml) MBC (,ug/ml) MIC (5Lg/ml) MBC (,g/ml)
1,024 64 2 32
> 1,024
>256 2 32
1,024 256 16 32
> 1,024. >256 32 32
calculation of the mean bacterial concentrations of the vegetations. For each therapy group, results are expressed as the mean ± standard deviation of the bacterial concentrations per gram of vegetation and as the number of sterile vegetations. The bacterial concentrations in the various groups were compared by an analysis of variance; this was followed by Fisher's test for multiple comparisons (20). The proportion of sterile vegetations of various groups was compared by the chi-square test. A P value of
Vol. 36, No. 12
0066-4804/92/122611-06$02.00/0 Copyright © 1992, American Society for Microbiology
Daptomycin or Teicoplanin in Combination with Gentamicin for Treatment of Experimental Endocarditis Due to a Highly Glycopeptide-Resistant Isolate of Enterococcus faecium FRANCOIS CARON,"* MARIE-DOMINIQUE KITZIS,2 LAURENT GUTMANN,2 ANNE-CLAUDE CREMIEUX,3 BERNARD MAZIERE,4 JEAN-MARIE VALLOIS,3 AZZAM SALEH-MGHIR,3 JEAN-FRAN§OIS LEMELAND,1 AND CLAUDE CARBON3 Groupe de Recherche en Infections Experimentales, H6pital Charles Nicolle, 76031 Rouen Cedex, 1 Laboratoire de Microbiologie Medicale, Universite Pierre et Marie Curie, 75270 Paris Cedex 06,2 Institut National de la Sante et de la Recherche Medicale, Unite 13, Hopital Bichat-Claude Bernard, 75944 Paris Cedex 19,3 and Service Hospitalier Frederic Joliot, Orsay,4 France Received 11 May 1992/Accepted 31 August 1992
Using an experimental endocarditis model, we studied the activity of daptomycin used alone or in combination with gentamicin against an Enterococcusfaecium strain that was highly resistant to glycopeptides and susceptible to gentamicin. In vitro, the MIC of daptomycin was 1 ,ug/ml. In vivo, daptomycin appeared to be effective only when it was used in a high-dose regimen, i.e., 12 mg/kg of body weight every 8 h (-2.5 log1o CFU/g versus controls; P < 0.05), particularly when it was combined with gentamicin (-5.0 log1o CFU/g versus controls; P < 0.01). Since the distribution of daptomycin into cardiac vegetations, as evaluated by autoradiography, appeared to be homogeneous, the poor in vivo activity of daptomycin was considered to be related to its high degree of protein binding, as suggested by killing curves studies. Since the MIC of teicoplanin for the vancomycin-resistant E.faecium strain used in the study was only 64 ,ug/ml and since an in vitro synergy between teicoplanin at high dose and gentamicin was observed, a high-dose regimen of teicoplanin, i.e., 40 mg/kg every 12 h, was also assessed in vivo. This treatment provided marginal activity only when it was combined with gentamicin (-2.3 log1o CFU/g versus controls; P < 0.05). These results suggest that the levels of daptomycin or teicoplanin in serum required to cure experimental endocarditis caused by a highly glycopeptide-resistant strain of E. faecium would not be achievable in humans.
Glycopeptide-resistant strains of enterococci, mainly Enterococcus faecium, have been observed with increasing frequency (4, 11). Most of the strains express high-level resistance to vancomycin (MICs, 64 to > 1,000 ,ug/ml) and, to a lesser extent, to teicoplanin (MICs, 32 to 256 ,ug/ml). The synergistic effect that vancomycin and aminoglycoside combinations usually display against enterococci in vitro is constantly abolished against vancomycin-resistant strains (13, 18). In the absence of cross-resistance between glycopeptide and lipopeptide antibiotics, daptomycin remains effective in vitro against such strains (4). Treatment of infections caused by highly glycopeptideresistant enterococci is not well defined. We have recently demonstrated in vitro and in an experimental endocarditis model the potential efficacy of the triple combination of penicillin-vancomycin-gentamicin for the treatment of serious infections caused by moderately penicillin- and highly glycopeptide-resistant enterococci (3). However, such a treatment could not be given to patients allergic to ,B-lactam antibiotics; in that case, other treatments are warranted. The first aim of the study described here was to evaluate the efficacy of daptomycin used alone or in combination with gentamicin for the treatment of experimental endocarditis caused by the clinical isolate of Enterococcusfaecium D400, which is highly resistant to glycopeptides and susceptible to gentamicin (3). The results obtained in animals were analyzed according to the in vitro activity of the drug, its pharmacokinetic parameters determined in animals, and a *
quantitative autoradiographic analysis evaluating its diffusion into cardiac vegetations. Since the MIC of teicoplanin for E. faecium D400 was only 64 ,ug/ml and since a synergy between teicoplanin at high concentration and gentamicin was observed in vitro, a second aim was to evaluate in vivo the efficacy of a highdose regimen of teicoplanin (HiD teicoplanin) and to compare it with the efficacy of a lower-dose regimen (LoD teicoplanin) which provides levels in animal sera comparable to those achieved in humans by the currently recommended regimen. MATERIALS AND METHODS Bacterial strain. E. faecium D400 (1, 3) was isolated from the stool of an immunocompromised patient who underwent digestive decontamination with vancomycin. Antibiotics and media. Daptomycin, teicoplanin, and gentamicin were kindly supplied, respectively, by Eli Lilly (Saint-Cloud, France), Marion Merrell Dow (LevalloisPerret, France), and Schering Plough (Levallois-Perret, France). In vitro studies were performed by using Mueller-Hinton broth (Diagnostics Pasteur, Marne-la-Coquette, France) supplemented with calcium (50 jig/ml) and magnesium (25 ,ug/ml) for daptomycin and by using brain heart infusion broth (Difco, Detroit, Mich.) for teicoplanin and gentamicin. Susceptibility tests. MICs and MBCs were determined by the macrodilution method (16). Each value was determined with a final log-phase inoculum of 5 x 105 CFU/ml. The MIC was defined as the lowest concentration of drug that inhib-
Corresponding author. 2611
2612
CARON ET AL.
ited growth, i.e., visible turbidity after 24 h of incubation at 37°C. The MBC was determined by subculturing 10 p,l from each clear tube on agar plates and was defined as the lowest concentration that reduced the number of viable organisms by at least 99.9%. Killing curves. Killing curves were determined for daptomycin, teicoplanin, and gentamicin, which were used alone or in different combinations. Each value was determined with bacteria in the exponential growth phase by using a final inoculum of 5 x 107 CFU/ml in order to approximate in vitro the high bacterial concentrations observed in cardiac vegetations. The antibiotic concentrations were also chosen to reflect the levels and the ratios achieved with the various regimens (see below) in rabbit serum. These concentrations were 10 and 40 ,ug/ml for daptomycin, 20 and 90 ,ug/ml for teicoplanin, and 4 p,g/ml for gentamicin. Killing curves were also determined in the presence of 50% rabbit serum at the same final concentrations of each drug given above. Samples were removed at 0, 3, 6, and 24 h in order to determine, by serial dilutions and plating in duplicate, the number of viable bacteria. Synergy was defined as a decrease in the CFU per milliliter by at least 2 log1o units compared with the effect of the most active single component in the drug combination. Experimental endocarditis. A bacterial aortic endocarditis was established by the technique of Perlman and Freedman (15), as follows. On day 1, female New Zealand White rabbits were anesthetized by intramuscular injections with ketamine hydrochloride (15 mg/kg of body weight). A carotid artery was exposed and cannulated with a polyethylene catheter. The catheter was advanced until pulsations and resistance indicated that it had reached the apex of the left ventricle. It was then sealed with a ligature and was left in place for the duration of the experiment. Twenty-four hours after surgery, animals were injected in the marginal vein with a log-phase inoculum of E. faecium D400 of approximately 108 CFU in a volume of 1 ml. A total of six rabbits receiving no treatment were used as controls. Eighty-four animals were treated from days 4 to 8, i.e., for 5 days, and were assigned to one of the following nine groups: (i) LoD daptomycin, i.e., 10 mg/kg twice daily (BID); (ii) HiD daptomycin, i.e., 12 mg/kg three times daily (TID); (iii) LoD teicoplanin, i.e., 10 mg/kg BID; (iv) HiD teicoplanin, i.e., 40 mg/kg BID; (v) gentamicin, 6 mg/kg BID; (vi) LoD daptomycin plus gentamicin; (vii) HiD daptomycin plus gentamicin; (viii) LoD teicoplanin plus gentamicin; and (ix) HiD teicoplanin plus gentamicin. The first dose of daptomycin or teicoplanin was doubled for loading. On day 9, animals were killed when each drug concentration reached a trough, i.e., 8 h after the last injection of the antibiotic given TID (i.e., for HiD daptomycin) or 12 h after the last injection of the antibiotics given BID (i.e., for the other drug regimens), and the heart was removed and inspected. Some rabbits were excluded because of perioperative death, negative pretreatment blood cultures, early death during the -course of treatment, or incorrect placement of the catheter across the aortic valve. All vegetations from each animal were excised, pooled, weighed, and rinsed in sterile saline. Vegetations from each animal were homogenized in 0.5 ml of sterile saline, and 0.1-ml aliquots were quantitatively subcultured on brain heart infusion agar plates at 37°C for 24 h. Portions of each vegetation homogenate were also cultured by the disk diffusion method to confirm the persistence of the initial pattern of antibiotic susceptibility of E. faecium D400 after the therapy period. The lower limit of detection by this method was 100 CFU/g. This value of 2 log1o CFU/g was assigned to all sterile vegetations and was used for the
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. In vitro antimicrobial susceptibility of E. faecium D400 determined in the absence or presence of rabbit serum Antibiotic
Vancomycin Teicoplanin Daptomycin Gentamicin
Medium without serum
Medium with 50% serum
MIC (,ug/ml) MBC (,ug/ml) MIC (5Lg/ml) MBC (,g/ml)
1,024 64 2 32
> 1,024
>256 2 32
1,024 256 16 32
> 1,024. >256 32 32
calculation of the mean bacterial concentrations of the vegetations. For each therapy group, results are expressed as the mean ± standard deviation of the bacterial concentrations per gram of vegetation and as the number of sterile vegetations. The bacterial concentrations in the various groups were compared by an analysis of variance; this was followed by Fisher's test for multiple comparisons (20). The proportion of sterile vegetations of various groups was compared by the chi-square test. A P value of
