ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1990, p. 2387-2391
Vol. 34, No. 12
0066-4804/90/122387-05$02.00/0 Copyright © 1990, American Society for Microbiology
Importance of the Aminoglycoside Dosing Regimen in the PenicillinNetilmicin Combination for Treatment of Enterococcus faecalisInduced Experimental Endocarditis BRUNO FANTIN AND CLAUDE CARBON* Institut National de la Sante et de la Recherche Medicale, Unite 13, H6pital Claude-Bernard, 75019 Paris, France Received 21 May 1990/Accepted 24 September 1990
The penicillin-aminoglycoside combination is recommended for the treatment of systemic enterococcal infections. However, the optimal dosing regimen of the aminoglycoside remains to be elucidated. We evaluated the efficacy of penicillin, alone or in combination with various dosing regimens of netilmicin, for the treatment of experimental left-sided Enterococcusfaecalis endocarditis in rabbits. Animals were injected intramuscularly for 4 days with penicillin alone or in combination with netilmicin in one of the following regimens: netilmicin at a low dose (2 mg/kg of body weight every 8 h), netilmicin at a high dose (4 mg/kg every 8 h), or netilmicin at a single daily high dose (12 mg/kg every 24 h). MICs and MBCs were 3.1 and 6.2 ,ug/ml and 8 and 8 ,ug/ml for penicillin and netilmicin, respectively. A netilmicin concentration of 4 ,ug/ml was the lowest concentration that achieved synergism with penicillin, as shown by the kill-curve method. Mean peak levels of netilmicin in serum were 5.6 (netilmicin at 2 mg/kg), 9.8 (netilmicin at 4 mg/kg), and 20.6 (netilmicin at 12 mg/kg) ,g/ml. Mean penicillin levels in serum were constantly above the MIC. Penicillin plus netilmicin at a high dose given three times daily was more effective (P < 0.05) than any other regimen in reducing bacterial titers in vegetations and was the only treatment that induced a significant bactericidal activity in rabbit serum during the trough. We concluded that divided doses of aminoglycoside are more effective than the same total dose given once daily in combination with penicillin. Our data suggest that prolonged levels of aminoglycoside in serum might be important to exhibit the greatest in vivo efficacy of the combination against E. faecalis. They also indicate that use of a reduced total daily dose of aminoglycoside or an increase in the interval between each dose might reduce the efficacy of therapy in animals with this type of infection.
Enterococci cause an estimated 10 to 20% of cases of bacterial endocarditis (18). As with other enterococcal infections, most isolates are Enterococcus faecalis (22). It is generally recognized that combinations of penicillin and aminoglycoside antibiotics are synergistic and, therefore, represent optimal therapy for endocarditis and other serious infections caused by enterococci (7, 22). Increasing numbers of enterococcal strains are becoming highly resistant to streptomycin (MIC, >2,000 p,g/ml), which results in a lack of synergistic killing when this agent is combined with penicillin (20, 28). However, combination of penicillin with gentamicin or netilmicin still results in bactericidal synergism against most E. faecalis strains which are highly resistant to streptomycin (21, 26). There is a close correlation between in vitro and in vivo results, with a clear advantage of penicillinaminoglycoside combinations (penicillin-gentamicin or penicillin-netilmicin) over penicillin alone against experimental endocarditis induced by streptomycin-susceptible or -resistant strains (3, 4, 15). The best dosing regimen for the treatment of enterococcal infections remains to be elucidated. For penicillin, sufficient data are available in the literature to suggest that antibiotic levels should be maintained above the MIC throughout the dosing interval, to prevent loss of efficacy as a result of bacterial regrowth, since no postantibiotic effect has been observed in vivo for penicillin, even in combination with an aminoglycoside, in the treatment of streptococcal or enterococcal infections in animals (12, 29). In contrast, the optimal *
dosing regimen for aminoglycosides in combination with penicillin remains controversial. Since the penicillin-aminoglycoside combination is recommended for a prolonged period of time in the treatment of enterococcal endocarditis, thus exposing the patient to the potential nephrotoxic and ototoxic side effects of aminoglycosides, the optimal aminoglycoside dosing regimen should provide maximal efficacy and minimal toxicity. In order to reduce aminoglycoside toxicity, some investigators suggested the use of low doses of gentamicin (30) or other aminoglycosides with lower toxicities, such as netilmicin (4, 15). This is the aminoglycoside of choice in our institution, precisely because of its lower toxicity (6, 13), and therefore was the aminoglycoside evaluated in the present experimental study. Another way to attenuate aminoglycoside toxicity would be to inject the antibiotic once daily, since this dosing regimen is well tolerated in humans (24, 25) and is less ototoxic and nephrotoxic than conventional dosing regimens in animal studies (1, 10, 25, 31). However, unlike the therapy of infections caused by gram-negative bacilli, the effectiveness of aminoglycosides given in a single daily dose has never been studied in vivo for the treatment of enterococcal infections. The aim of this study was to evaluate in vitro the bactericidal activity of the penicillin-netilmicin combination against a clinical strain of E. faecalis and to evaluate in vivo, using the experimental endocarditis model, the efficacy of this combination with a fixed penicillin regimen but various aminoglycoside dosing regimens: a high dose three times daily, the same total daily dose given once daily, and a low dose given three times daily.
Corresponding author. 2387
2388
FANTIN AND CARBON
MATERIALS AND METHODS
ANTIMICROB. AGENTS CHEMOTHER.
37°C for 24 h. Colony counts are expressed as log1o CFU per
gram of vegetation.
Bacterial strain. The study strain was an E. faecalis organism isolated from the blood of a patient with mediastinitis and septicemia. This strain was inhibited by 2,000 ,ug of streptomycin per ml. Antibiotics. Penicillin was supplied by Diamant Laboratories (Montrouge, France); gentamicin and netilmicin were supplied by Unilabo Laboratories (Levallois-Perret, France). Susceptibility studies. MICs and MBCs were determined by the microdilution method, with an inoculum of 106 CFU/ ml, in Mueller-Hinton broth supplemented with Ca2' and Mg2+ (Diagnostic Pasteur, Marnes-la-Coquette, France). Each value was determined in duplicate. The MBC was determined by subculturing 0.01-ml samples onto MuellerHinton agar plates containing 106 U of penicillinase, and the plates were incubated at 37°C. After 24 h, colonies were counted. The MBC was defined as the lowest concentration of drug that reduced the number of viable organisms by 99.9% (23). In vitro synergy tests. Bactericidal rates were determined in Mueller-Hinton broth by using an inoculum of 106 CFU/ml while the organism was in the log phase of growth. The following concentrations were used: 2.5, 5, 10, or 20 U of penicillin alone per ml; 4, 8, or 20 ,ug of netilmicin alone per ml; or 5U of penicillin in combination with 2, 4, 8, or 20 ,ug of netilmicin per ml. After 0, 4, and 24 h of incubation at 37°C, serial dilutions of 0.1-ml samples (to 10-6) were subcultured in duplicate onto agar plates containing 106 U of penicillinase and were incubated at 37°C for 24 h. Synergy was defined as at least a 100-fold increase in killing after 24 h of incubation in the presence of a combination of antibiotics compared with that achieved by the most effective agent alone (16). Experimental endocarditis. Investigations were performed in 38 female New Zealand White rabbits (weight range, 1.8 to 2.1 kg). Aortic endocarditis was induced as described previously (8, 9). Twenty-four hours after catheter insertion, 1 ml containing 108 E. faecalis CFU/ml was injected into an ear vein. Seventy-two hours after inoculation, animals received intramuscularly one of the following regimens for 4 days: penicillin alone (penicillin G [400,000 U] plus procaine penicillin G [600 000 U]) twice daily (8 a.m., 8 p.m.), penicillin plus netilmicin at a low dose given as 2 mg/kg of body weight every 8 h (8 a.m., 4 p.m., 12 p.m.), or penicillin plus netilmicin at a high dose (12 mg/kg/day) given either as 4 mg/kg every 8 h (8 a.m., 4 p.m., 12 p.m.) or as a single daily dose at 8 a.m. For penicillin, the regimen was chosen to achieve levels in serum that were constantly above the MIC. For netilmicin, the 4-mg/kg regimen reproduced peak levels in serum comparable to those obtained in humans. A control group was left untreated. On day 4 of antimicrobial therapy, blood was sampled 1 h after the 8 a.m. injection and at the time of sacrifice (i.e., 8, 12, and 24 h after the last antibiotic dose for netilmicin given every 8 h and for penicillin and netilmicin given every 24 h, respectively), for determination of peak and trough antibiotic levels and bactericidal titers in serum. Animals were killed by intravenous injection of pentobarbital. At the time of sacrifice, the heart was removed and the chambers on the left side were examined to confirm vegetative endocarditis. Each vegetation was excised, weighed, and homogenized in 0.5 ml of sterile saline, and 0.1-ml portions were quantitatively subcultured onto agar plates containing penicillinase and were incubated at
Antibiotic assays. Antibiotic concentrations in serum were measured by the agar diffusion method, using Bacillus subtilis ATCC 6633 for penicillin and netilmicin assays, as described previously (8). For netilmicin assays, 106 U of penicillinase per ml of serum was added. Penicillin assays were performed in serum from rabbits that received penicillin alone. The lower limits of sensitivity were 0.1 and 0.5 ,ug/ml for netilmicin and penicillin, respectively. Bactericidal titer in serum. Bactericidal titers in serum were determined in a final volume of 100 RI in 50% MuellerHinton broth with an inoculum of 106 CFU/ml (27). Serial twofold dilutions of serum were made (range, 1/2 to 1/256). After a 24-h incubation at 37°C, 0.01-ml aliquots were removed from all tubes and were subcultured onto MuellerHinton agar plates containing penicillinase. After 24 h of incubation at 37°C, the number of viable CFU was determined. The bactericidal titer in serum was defined as the highest dilution that killed at least 99.9% of the original inoculum (23). Statistics. The bacterial concentrations in vegetations from the various groups of animals were compared by an analysis of variance followed by the Scheffe test for multiple comparisons. A P value of
Vol. 34, No. 12
0066-4804/90/122387-05$02.00/0 Copyright © 1990, American Society for Microbiology
Importance of the Aminoglycoside Dosing Regimen in the PenicillinNetilmicin Combination for Treatment of Enterococcus faecalisInduced Experimental Endocarditis BRUNO FANTIN AND CLAUDE CARBON* Institut National de la Sante et de la Recherche Medicale, Unite 13, H6pital Claude-Bernard, 75019 Paris, France Received 21 May 1990/Accepted 24 September 1990
The penicillin-aminoglycoside combination is recommended for the treatment of systemic enterococcal infections. However, the optimal dosing regimen of the aminoglycoside remains to be elucidated. We evaluated the efficacy of penicillin, alone or in combination with various dosing regimens of netilmicin, for the treatment of experimental left-sided Enterococcusfaecalis endocarditis in rabbits. Animals were injected intramuscularly for 4 days with penicillin alone or in combination with netilmicin in one of the following regimens: netilmicin at a low dose (2 mg/kg of body weight every 8 h), netilmicin at a high dose (4 mg/kg every 8 h), or netilmicin at a single daily high dose (12 mg/kg every 24 h). MICs and MBCs were 3.1 and 6.2 ,ug/ml and 8 and 8 ,ug/ml for penicillin and netilmicin, respectively. A netilmicin concentration of 4 ,ug/ml was the lowest concentration that achieved synergism with penicillin, as shown by the kill-curve method. Mean peak levels of netilmicin in serum were 5.6 (netilmicin at 2 mg/kg), 9.8 (netilmicin at 4 mg/kg), and 20.6 (netilmicin at 12 mg/kg) ,g/ml. Mean penicillin levels in serum were constantly above the MIC. Penicillin plus netilmicin at a high dose given three times daily was more effective (P < 0.05) than any other regimen in reducing bacterial titers in vegetations and was the only treatment that induced a significant bactericidal activity in rabbit serum during the trough. We concluded that divided doses of aminoglycoside are more effective than the same total dose given once daily in combination with penicillin. Our data suggest that prolonged levels of aminoglycoside in serum might be important to exhibit the greatest in vivo efficacy of the combination against E. faecalis. They also indicate that use of a reduced total daily dose of aminoglycoside or an increase in the interval between each dose might reduce the efficacy of therapy in animals with this type of infection.
Enterococci cause an estimated 10 to 20% of cases of bacterial endocarditis (18). As with other enterococcal infections, most isolates are Enterococcus faecalis (22). It is generally recognized that combinations of penicillin and aminoglycoside antibiotics are synergistic and, therefore, represent optimal therapy for endocarditis and other serious infections caused by enterococci (7, 22). Increasing numbers of enterococcal strains are becoming highly resistant to streptomycin (MIC, >2,000 p,g/ml), which results in a lack of synergistic killing when this agent is combined with penicillin (20, 28). However, combination of penicillin with gentamicin or netilmicin still results in bactericidal synergism against most E. faecalis strains which are highly resistant to streptomycin (21, 26). There is a close correlation between in vitro and in vivo results, with a clear advantage of penicillinaminoglycoside combinations (penicillin-gentamicin or penicillin-netilmicin) over penicillin alone against experimental endocarditis induced by streptomycin-susceptible or -resistant strains (3, 4, 15). The best dosing regimen for the treatment of enterococcal infections remains to be elucidated. For penicillin, sufficient data are available in the literature to suggest that antibiotic levels should be maintained above the MIC throughout the dosing interval, to prevent loss of efficacy as a result of bacterial regrowth, since no postantibiotic effect has been observed in vivo for penicillin, even in combination with an aminoglycoside, in the treatment of streptococcal or enterococcal infections in animals (12, 29). In contrast, the optimal *
dosing regimen for aminoglycosides in combination with penicillin remains controversial. Since the penicillin-aminoglycoside combination is recommended for a prolonged period of time in the treatment of enterococcal endocarditis, thus exposing the patient to the potential nephrotoxic and ototoxic side effects of aminoglycosides, the optimal aminoglycoside dosing regimen should provide maximal efficacy and minimal toxicity. In order to reduce aminoglycoside toxicity, some investigators suggested the use of low doses of gentamicin (30) or other aminoglycosides with lower toxicities, such as netilmicin (4, 15). This is the aminoglycoside of choice in our institution, precisely because of its lower toxicity (6, 13), and therefore was the aminoglycoside evaluated in the present experimental study. Another way to attenuate aminoglycoside toxicity would be to inject the antibiotic once daily, since this dosing regimen is well tolerated in humans (24, 25) and is less ototoxic and nephrotoxic than conventional dosing regimens in animal studies (1, 10, 25, 31). However, unlike the therapy of infections caused by gram-negative bacilli, the effectiveness of aminoglycosides given in a single daily dose has never been studied in vivo for the treatment of enterococcal infections. The aim of this study was to evaluate in vitro the bactericidal activity of the penicillin-netilmicin combination against a clinical strain of E. faecalis and to evaluate in vivo, using the experimental endocarditis model, the efficacy of this combination with a fixed penicillin regimen but various aminoglycoside dosing regimens: a high dose three times daily, the same total daily dose given once daily, and a low dose given three times daily.
Corresponding author. 2387
2388
FANTIN AND CARBON
MATERIALS AND METHODS
ANTIMICROB. AGENTS CHEMOTHER.
37°C for 24 h. Colony counts are expressed as log1o CFU per
gram of vegetation.
Bacterial strain. The study strain was an E. faecalis organism isolated from the blood of a patient with mediastinitis and septicemia. This strain was inhibited by 2,000 ,ug of streptomycin per ml. Antibiotics. Penicillin was supplied by Diamant Laboratories (Montrouge, France); gentamicin and netilmicin were supplied by Unilabo Laboratories (Levallois-Perret, France). Susceptibility studies. MICs and MBCs were determined by the microdilution method, with an inoculum of 106 CFU/ ml, in Mueller-Hinton broth supplemented with Ca2' and Mg2+ (Diagnostic Pasteur, Marnes-la-Coquette, France). Each value was determined in duplicate. The MBC was determined by subculturing 0.01-ml samples onto MuellerHinton agar plates containing 106 U of penicillinase, and the plates were incubated at 37°C. After 24 h, colonies were counted. The MBC was defined as the lowest concentration of drug that reduced the number of viable organisms by 99.9% (23). In vitro synergy tests. Bactericidal rates were determined in Mueller-Hinton broth by using an inoculum of 106 CFU/ml while the organism was in the log phase of growth. The following concentrations were used: 2.5, 5, 10, or 20 U of penicillin alone per ml; 4, 8, or 20 ,ug of netilmicin alone per ml; or 5U of penicillin in combination with 2, 4, 8, or 20 ,ug of netilmicin per ml. After 0, 4, and 24 h of incubation at 37°C, serial dilutions of 0.1-ml samples (to 10-6) were subcultured in duplicate onto agar plates containing 106 U of penicillinase and were incubated at 37°C for 24 h. Synergy was defined as at least a 100-fold increase in killing after 24 h of incubation in the presence of a combination of antibiotics compared with that achieved by the most effective agent alone (16). Experimental endocarditis. Investigations were performed in 38 female New Zealand White rabbits (weight range, 1.8 to 2.1 kg). Aortic endocarditis was induced as described previously (8, 9). Twenty-four hours after catheter insertion, 1 ml containing 108 E. faecalis CFU/ml was injected into an ear vein. Seventy-two hours after inoculation, animals received intramuscularly one of the following regimens for 4 days: penicillin alone (penicillin G [400,000 U] plus procaine penicillin G [600 000 U]) twice daily (8 a.m., 8 p.m.), penicillin plus netilmicin at a low dose given as 2 mg/kg of body weight every 8 h (8 a.m., 4 p.m., 12 p.m.), or penicillin plus netilmicin at a high dose (12 mg/kg/day) given either as 4 mg/kg every 8 h (8 a.m., 4 p.m., 12 p.m.) or as a single daily dose at 8 a.m. For penicillin, the regimen was chosen to achieve levels in serum that were constantly above the MIC. For netilmicin, the 4-mg/kg regimen reproduced peak levels in serum comparable to those obtained in humans. A control group was left untreated. On day 4 of antimicrobial therapy, blood was sampled 1 h after the 8 a.m. injection and at the time of sacrifice (i.e., 8, 12, and 24 h after the last antibiotic dose for netilmicin given every 8 h and for penicillin and netilmicin given every 24 h, respectively), for determination of peak and trough antibiotic levels and bactericidal titers in serum. Animals were killed by intravenous injection of pentobarbital. At the time of sacrifice, the heart was removed and the chambers on the left side were examined to confirm vegetative endocarditis. Each vegetation was excised, weighed, and homogenized in 0.5 ml of sterile saline, and 0.1-ml portions were quantitatively subcultured onto agar plates containing penicillinase and were incubated at
Antibiotic assays. Antibiotic concentrations in serum were measured by the agar diffusion method, using Bacillus subtilis ATCC 6633 for penicillin and netilmicin assays, as described previously (8). For netilmicin assays, 106 U of penicillinase per ml of serum was added. Penicillin assays were performed in serum from rabbits that received penicillin alone. The lower limits of sensitivity were 0.1 and 0.5 ,ug/ml for netilmicin and penicillin, respectively. Bactericidal titer in serum. Bactericidal titers in serum were determined in a final volume of 100 RI in 50% MuellerHinton broth with an inoculum of 106 CFU/ml (27). Serial twofold dilutions of serum were made (range, 1/2 to 1/256). After a 24-h incubation at 37°C, 0.01-ml aliquots were removed from all tubes and were subcultured onto MuellerHinton agar plates containing penicillinase. After 24 h of incubation at 37°C, the number of viable CFU was determined. The bactericidal titer in serum was defined as the highest dilution that killed at least 99.9% of the original inoculum (23). Statistics. The bacterial concentrations in vegetations from the various groups of animals were compared by an analysis of variance followed by the Scheffe test for multiple comparisons. A P value of
