Effect of Gentamicin Dosing Interval on Therapy of Viridans Streptococcal Experimental Endocarditis with Gentamicin plus Penicillin

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1995, p. 2098 2103 Vol. 39, No. 9 0066-4804/95/$04.00 0 Copyright 1995, American Society for Microbiology Effect of Gentamicin Dosing Interval on Therapy of Viridans Streptococcal Experimental Endocarditis with Gentamicin plus Penicillin JOAN GAVALDÀ, 1 * ALBERT PAHISSA, 1 BENITO ALMIRANTE, 1 MONTSERRAT LAGUARDA, 1 ERNESTO CRESPO, 2 LEONOR POU, 3 AND FRANCISCO FERNÁNDEZ 2 Infectious Diseases Research Laboratory, Infectious Diseases Division, 1 and Biochemistry 3 and Microbiology 2 Services, Hospital Universitari Vall d Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain Received 6 May 1994/Returned for modification 16 December 1994/Accepted 16 May 1995 This study compares the effects of a total daily dose of gentamicin given once a day (q.d.) or three times a day (t.i.d.) in the therapy of experimental endocarditis in rabbits caused by penicillin-susceptible, penicillintolerant, or penicillin-resistant viridans streptococci. Four isolates were used in vivo: one penicillin susceptible (MIC < 0.03 g/ml), one penicillin tolerant (MBC/MIC, <0.03/>32 g/ml), and two penicillin resistant (MICs 0.5 and 2 g/ml). Animals were infected with one of the four isolates and assigned to one of the following treatment regimens: no treatment, procaine penicillin at 1.2 million IU intramuscularly (i.m.) t.i.d., procaine penicillin plus gentamicin at 1 mg/kg of body weight i.m. t.i.d., procaine penicillin plus gentamicin at 3 mg/kg i.m. q.d., or procaine penicillin plus gentamicin at 1 mg/kg i.m. q.d. (only animals infected with the penicillinsusceptible isolate). Serum drug concentrations measured 30 min after administration of 1.2 million IU of penicillin and 1 or 3 mg of gentamicin per kg were 22.6, 3.8, and 8.5 g/ml, respectively. The reduced total daily dose of gentamicin was ineffective among animals infected with penicillin-susceptible viridans streptococci; treatment with 1 mg of gentamicin per kg per day plus penicillin was less effective (P < 0.05) than was treatment with 3 mg of gentamicin per kg per day plus penicillin. The 1-mg/kg/day gentamicin treatment regimen was not further studied. The gentamicin dosing interval did not significantly affect (q.d. versus t.i.d., P > 0.05) the relative efficacy of penicillin plus gentamicin for treatment of experimental endocarditis among animals infected with each of the four isolates tested. * Corresponding author. Mailing address: Infectious Diseases Division, Hospital General Universitari Vall d Hebron, Avda. Vall d Hebron, 119-129, 08035 Barcelona, Spain. Phone: 34.3.4282637. Fax: 34.3.4282637. Electronic mail address: J_gavalda@ar.vhebron.es. A 2- to 6-weeks course of therapy with a -lactam plus an aminoglycoside antibiotic, depending on the patient and the species and susceptibility of the infecting organism, is effective treatment for streptococcal endocarditis (5). Because of the potential nephrotoxic effects of prolonged aminoglycoside therapy and the prolonged hospitalization period required for these patients, equally effective, less-toxic treatment regimens amenable to home health care may be therapeutic options offering more benefits and fewer risks to the patient. Various approaches to reducing aminoglycosideassociated nephrotoxicity while maintaining clinical efficacy have been investigated and include reducing the total daily dose, use of possibly less-toxic agents, and increasing the dosing interval for aminoglycoside administration. Increasing the dosing interval has been shown to be no more toxic than is intermittent administration (3, 7, 11, 18, 21, 30, 33 37, 41 43). The efficacy of this approach has been tested with experimental gram-negative rod infections (9, 19, 21, 24 26, 31, 43), penicillin-susceptible (6, 16, 38) or -resistant (6, 16) viridans streptococcal and enterococcal (15) experimental endocarditis, and human infections other than endocarditis (4, 14, 20, 23, 27, 36, 39, 40). However, although there is one study (38) in which once-a-day treatment with penicillin plus an aminoglycoside was compared with the standard treatment (penicillin plus an aminoglycoside in three divided doses) for endocarditis caused by penicillin-susceptible streptococci, this issue has not been evaluated to date in the treatment of experimental endocarditis caused by penicillin-tolerant or -resistant viridans streptococci. The aim of this study was to compare the efficacy of the standard therapeutic regimen (penicillin plus gentamicin at 1 mg/kg of body weight to reach a peak concentration in serum of 3 g/ml, every 8 h) with that of a regimen of penicillin plus the same total daily dose of gentamicin (3 mg/kg) given at an increased dosing interval, once a day, for experimental endocarditis caused by four different viridans streptococcal strains which encompass the main antibiotic susceptibility phenotypes encountered in clinics (penicillin susceptible, penicillin tolerant, and moderately or highly penicillin resistant). Likewise, we have evaluated the efficacy of a low dose of gentamicin (1 mg/kg) given once a day in combination with penicillin in the treatment of penicillin-susceptible viridans streptococcal experimental endocarditis. (This work was presented in part at the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif., October 1992 [17a].) MATERIALS AND METHODS In vitro studies. (i) Microorganisms. Four viridans streptococcal blood isolates with different susceptibilities to penicillin recovered from patients with endocarditis seen at our hospital were used: one penicillin-susceptible strain (PSVS), one tolerant strain (PTVS), and two resistant strains (PrVS and PRVS). The PrVS strain was identified as Streptococcus intermedius, the PRVS strain was identified as Streptococcus sanguis I, and the remaining two were identified as S. sanguis II. Neither of these strains was nutritionally variant. Working stock cultures were stored in skim milk at 80 C. Before each experiment, an aliquot was thawed and subcultured onto 5% sheep blood Columbia agar plates. (ii) Susceptibility studies. MICs and MBCs were determined in duplicate by the tube macrodilution method. Inocula were prepared from logarithmic-phase cultures to yield a final concentration of 10 5 CFU/ml and inoculated into serial twofold dilutions of penicillin or gentamicin in Mueller-Hinton broth supplemented with 3% lysed sheep blood (2). Subcultures were used for confirmation 2098

VOL. 39, 1995 GENTAMICIN AND STREPTOCOCCAL EXPERIMENTAL ENDOCARDITIS 2099 of purity and quantification of inoculum size. Tubes containing the inocula in serial dilutions of penicillin or gentamicin were incubated for 24 h at 35 C in room air. The MIC was defined as the lowest concentration of antimicrobial agent that did not allow visible growth. The MBC was determined from subcultures onto 5% sheep blood Columbia agar plates of 0.05 ml each from the control tube, from the first tube with visible growth, and from all tubes without visible growth. The plates were incubated for 72 h at 35 C at room temperature. The MBC was defined as the lowest concentration of antibiotic that killed 99.9% of the original inoculum (2). Tolerance was defined as an MBC/MIC ratio of 32. (iii) Studies of antimicrobial synergy. Tests for in vitro synergy were performed by the time-kill method. Killing curves were determined by using Mueller-Hinton broth supplemented with 3% sheep blood with an inoculum size of 10 6 CFU/ml in the logarithmic growth phase for the four strains used for animal experimental endocarditis. The following concentrations of antibiotics, both alone and in combination, were used: penicillin at one-half the MIC and gentamicin at 1, 3, and 6 g/ml. After 0, 3, 6, and 24 h of incubation at 35 C, serial dilutions of 0.1-ml samples (to 10 6 ) were subcultured onto 5% sheep blood Columbia agar plates and incubated for 72 h at 35 C in room air. Each experiment was run twice in duplicate and included controls of growth without antibiotic. In vitro synergy was defined as at least a 2-log-unit increase in killing after 24 h of incubation in the presence of a combination of antibiotics compared with the level achieved by the most effective agent alone (13). Serum pharmacokinetic studies. To determine concentrations of antibiotic in serum, 1 ml of blood was drawn from the ear artery of each rabbit at 0.5, 1, 2, 4, 6, 8, and 12 h after intramuscular (i.m.) injection of 1.2 million IU of procaine penicillin and 1 or 3 mg of gentamicin per kg. Groups of four healthy rabbits were used for procaine penicillin treatment, and five healthy animals were used for each gentamicin dosage regimen. Blood samples were placed into glass tubes and centrifuged (10 min, 1,500 g). The serum was stored at 80 C, and all the samples were assayed on the same day. Penicillin concentrations were determined by the disk-plate bioassay method (1) with Micrococcus luteus ATCC 9341 as the bioassay microorganism and Antibiotic Medium 5 (Difco Laboratories, Detroit, Mich.) as the growth medium. Standard curves were determined with solutions of penicillin (0.1, 0.2, 0.3, 0.4, and 0.5 g/ml) in pooled rabbit serum. The concentrations in serum were derived from the standard curve. The serum samples from the rabbits were diluted in pooled rabbit serum so that the concentration of drug would be within the range of the standard curve. The standard samples were assayed in quintuplicate, and the serum samples were assayed in triplicate. Results were expressed as micrograms per milliliter of blood. The linearity (r 2 ) of the standard curve was 0.995. The sensitivity of the assay was about 0.1 g/ml of sample, and the coefficients of between- and within-day variation for replicates (n 7) at 0.1 and 30 g/ml were 9.6 and 4.97% and 5.23 and 3.62%, respectively. Concentrations of gentamicin in serum were measured by fluorescence polarization (TDx; Abbott Diagnostics, Irving, Tex.) according to the manufacturer s directions. The sensitivity of the assay was about 0.25 g/ml of sample, and the coefficients of between- and within-day variation for replicates (n 10) at 1.22 and 3.92 g/ml were 8.9 and 6.7% and 4 and 3.5%, respectively. The half-lives at phase of the drugs were calculated as ln 2/, where is the elimination rate constant. The elimination rate constant was determined as the slope obtained from a linear regression analysis of the plasma concentrationversus-time curve on the basis of an open one-compartment model. In vivo studies. Experimental aortic valve infective endocarditis was established in New Zealand White rabbits (weight approximately 2 to 2.6 kg) by modifications of the method described previously by Garrison and Freedman (17) and Perlman and Freedman (32) and modified by Durack and Beeson (12). Briefly, animals were anesthetized with ketamine at 100 mg/kg and xylazene at 20 mg/kg, injected i.m. A midline incision was made in the neck, and the right carotid artery was exposed. The artery was ligated distally, and a sterile polyethylene catheter with an internal diameter of 0.76 mm and an external diameter of 1.22 mm (PE 90; Clay Adams, Becton Dickinson, Parsippany, N.J.) was inserted into the artery through a small incision and advanced proximally across the aortic valve into the left ventricle. Proper positioning of the catheter tip inside the left ventricle was achieved with a pressure-sensitive monitoring device attached to the distal end of the catheter. The catheter was secured with silk sutures, sealed, tied off to the carotid artery, and left in place for the duration of the experiment. The wound was closed over the catheter with a continuous silk suture. Different groups of animals were infected with one of the strains (PSVS, PTVS, PrVS, and PRVS). Viridans streptococci were inoculated into tryptic soy broth, and cultures were incubated overnight and diluted to the desired concentration. Twenty-four hours after the placement of the intracardiac catheter, 1 ml of saline containing 10 5 CFU of PSVS, PTVS, or PrVS per ml was injected into a peripheral ear vein. Animals with endocarditis caused by PRVS were infected with 10 6 CFU/ml in 1 ml of saline. A 1-ml volume of blood was obtained after infection and just before initiation of antimicrobial therapy in order to confirm the presence of endocarditis. The blood specimen was mixed with 20 ml of molten tryptic soy agar. Plates were incubated for 48 h at 35 C in room air, and the presence of viridans streptococci was interpreted as indicative of infective endocarditis. Antimicrobial therapy was initiated 48 h after infection with the strains PSVS, PTVS, and PrVS and 72 h after intravenous injection of the PRVS strain. Animals with endocarditis caused by PRVS were infected with a larger inoculum and treated after a longer period of infection because of the lower virulence of TABLE 1. MICs and MBCs of penicillin and gentamicin against the four strains used in this study MIC/MBC ( g/ml) Strain Penicillin Gentamicin PSVS, S. sanguis II 0.03/ 0.03 8/16 PTVS, S. sanguis II 0.03/ 32 8/16 PrVS, S. intermedius 0.5/8 8/8 PRVS, S. sanguis I 2/2 8/16 this strain. Antimicrobial agents were administered i.m. for 3 days. Animals infected were placed in the following treatment groups: (i) control without treatment, (ii) penicillin G procaine at 1.2 million U every 8 h, (iii) penicillin at the same dosage plus gentamicin at 1 mg/kg i.m. every 8 h, (iv) penicillin at the same dosage plus gentamicin at 3 mg/kg i.m. once a day, and (v) penicillin at the same dosage plus gentamicin at 1 mg/kg i.m. once a day (only for animals with endocarditis caused by the penicillin-susceptible strain). After 3 days of treatment, animals were sacrificed by a lethal intravenous injection of sodium pentothal, at least 12 h after the last dose of antibiotic(s) was administered. The chest was opened, the heart was excised and opened, and the chambers of the left side were examined to confirm the presence of infective endocarditis. Only animals with proper placement of the catheter and macroscopic evidence of vegetations at the time of sacrifice were included in the study. The aortic valve vegetations were removed, pooled, weighed, and homogenized in 2 ml of tryptic soy broth in a tissue homogenizer (Stomacher 80 r ). The homogenates were serially diluted and quantitatively cultured by a pour plate method on tryptic soy broth supplemented with 5% defibrinated sheep blood and 1,000 IU of penicillinase. The plates were incubated for 48 h at 35 C in room air. Results were expressed as the log 10 CFU of viridans streptococci per gram of vegetation. Bacterial densities in valvular vegetations calculated to be between 0 and 2 log CFU/g were reported as 2 log 10 CFU/g rather than 0 because of potential errors associated with the low weight of the valvular tissue. Analysis of results. Results were expressed as the mean standard deviation, median, and range of log 10 CFU of viridans streptococci per gram of vegetation. Differences in log 10 CFU of viridans streptococci per gram of vegetations in the different therapy groups of the animals infected with the same strain were analyzed statistically by the Kruskall-Wallis test and Wilcoxon Mann-Whitney U rank sum analysis. The results were corrected for multiple comparisons (8). A P value of less than 0.05 was considered significant. RESULTS In vitro studies. The MICs and MBCs of penicillin and gentamicin for the four strains used are shown in Table 1. Neither of the penicillin-resistant strains has the trait of penicillin tolerance. The in vitro killing of the four strains of viridans streptococci by penicillin and gentamicin, both singly and in combination, is shown in Fig. 1 and 2. There was synergism and a reduction of at least 3 log 10 CFU/ml from the original inoculum after 24 h of incubation with the combination of penicillin and 3 or 6 g of gentamicin per ml against the PSVS, PrVS, and PRVS strains. The combination of penicillin with the lowest concentration of gentamicin (1 g/ml) was only synergistic against the PSVS strain. The time-kill curve for the PSVS, PrVS, and PRVS strains shows that gentamicin at the highest concentration tested (6 g/ml) combined with penicillin had an increased rate of killing in the first hours of incubation compared with the rate achieved with the combinations of penicillin and gentamicin at 1 or 3 g/ml. The tolerant strain was not inhibited by penicillin alone, and the combination of penicillin and gentamicin was only bacteriostatic (the culture of the PTVS strain had barely lost 1 log unit of viable counts after 24 h of incubation with the combination). The combinations of penicillin and gentamicin were synergistic on the basis of a definition for synergy of a decrease of 2 log CFU/ml compared with the result obtained with penicillin alone at 24 h of incubation. This occurred only because there was growth of the PTVS strain to 10 7 CFU/ml with penicillin alone.

2100 GAVALDÀ ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 1. In vitro killing rates for PSVS (A) and PTVS (B) incubated with penicillin at one-half the MIC and different regimens of gentamicin, both alone and in combination with penicillin. ç, control; F, penicillin;, gentamicin at 1 g/ml;, gentamicin at 3 g/ml;, gentamicin at 6 g/ml;, penicillin plus gentamicin at 1 g/ml; â, penicillin plus gentamicin at 3 g/ml; Ç É, penicillin plus gentamicin at 6 g/ml. FIG. 2. In vitro killing rates for PrVS (A) and PRVS (B) incubated with penicillin at one-half the MIC and different regimens of gentamicin, both alone and in combination with penicillin. ç, control; F, penicillin;, gentamicin at 1 g/ml;, gentamicin at 3 g/ml;, gentamicin at 6 g/ml;, penicillin plus gentamicin at 1 g/ml; â, penicillin plus gentamicin at 3 g/ml; Ç É, penicillin plus gentamicin at 6 g/ml. Serum pharmacokinetic studies. Antibiotic levels in serum of treated animals and elimination half-lives of penicillin and gentamicin are shown in Table 2. Mean serum penicillin levels were always above the MIC for the four strains used in the study. The peak serum drug concentrations in the animals were very similar to those expected to occur in humans after standard doses of penicillin and gentamicin. In vivo therapeutic studies. Results of therapy of experimental endocarditis caused by PSVS are shown in Table 3. After 3 days of treatment, residual bacterial titers in cardiac vegetation were significantly lower in treated animals than in controls (P 0.001). Penicillin plus a low dose (1 mg/kg) of gentamicin given once a day had an effect similar to that of penicillin alone (P 0.05) and was less effective than was penicillin combined with gentamicin at 3 mg/kg, both given in three divided doses and once a day (P 0.05). Gentamicin at 1 mg/kg every 8hor at 3 mg/kg once a day combined with penicillin was more effective than penicillin alone (P 0.001). Regimens with gentamicin at 3 mg/kg had similar efficacies whether given in three divided doses or once a day (P 0.05).

VOL. 39, 1995 GENTAMICIN AND STREPTOCOCCAL EXPERIMENTAL ENDOCARDITIS 2101 Time TABLE 2. Results of pharmacokinetic studies a Penicillin Serum drug concn ( g/ml) (mean SD) In spite of the fact that PTVS and PSVS behaved very differently in the in vitro time-kill studies, the therapeutic results in vivo were very similar: gentamicin at 3 mg/kg given once a day combined with penicillin had an efficacy similar to that of gentamicin given in three divided doses combined with penicillin (P 0.05), and both regimens were more effective than was penicillin alone (P 0.001) in the treatment of both PTVS and PSVS experimental endocarditis (Table 3). In the treatment of experimental endocarditis caused by resistant strains (Table 4), penicillin plus gentamicin at 3 mg/kg once a day was effective independently of the MIC/MBC ratio for isolates, with activity similar to that of the gold standard, penicillin plus gentamicin at 1 mg/kg three times a day (P 0.05). Also, penicillin plus gentamicin once or three times daily was more efficacious than was penicillin alone (P 0.01) in the treatment of PrVS experimental endocarditis. DISCUSSION Gentamicin at 1 mg/kg Gentamicin at 3 mg/kg 30 min 22.6 2.48 3.85 0.09 8.53 0.5 60 min 9.8 1.52 3.02 0.36 6.51 1.3 2 h 7.5 0.85 1.34 0.29 2.85 1.6 4 h 6.3 1.13 0.36 0.12 0.6 0.4 6 h 4.5 0.64 ND b ND 8 h 3.1 0.6 0.25 0.25 12 h 1.9 0.55 a Results show serum drug concentrations in rabbits after administration of procaine penicillin at 1.2 million IU i.m. or gentamicin at 1 or 3 mg/kg i.m. Half-lives at phase (in hours, with standard deviations) were as follows: penicillin, 3.95 0.55; gentamicin at both 1 and 3 mg/kg, 0.9 0.1. b ND, not done. In the studies described here, we explored whether penicillin in combination with gentamicin given once a day was as effective as was the same total dose of gentamicin administered in three divided doses in the treatment of experimental endocarditis caused by penicillin-susceptible, -tolerant, and -resistant viridans group streptococci. Studies conducted with the animal model of experimental endocarditis show that regimens with aminoglycosides once a day show an efficacy similar to that of those with aminoglycosides given in divided daily doses (6, 16, 38), except with experimental endocarditis caused by Enterococcus faecalis (15). This study demonstrated that both the total daily dose and the interval between doses are important parameters in determining the optimal aminoglycoside dosing regimen for treatment of enterococcal endocarditis. For the same total daily dose, an increased interval between aminoglycoside doses is associated with a loss of efficacy (15). Our results with the penicillin-susceptible viridans streptococcus model concur with those of Saleh-Mghir et al. (38); using a model of nutritionally variant viridans streptococcus experimental endocarditis, they showed that regimens of tobramycin at a dose of 3 mg/kg had similar efficacies whether given once daily (peak concentration in serum, 5.4 g/ml) or three times daily (peak concentration in serum, 2.4 g/ml) in combination with penicillin. Blatter et al. (6) and Francioli and Glauser (16) showed successful results using the combination of ceftriaxone and netilmicin in the treatment of endocarditis caused by penicillin-susceptible streptococci. The efficacy of gentamicin given once a day combined with penicillin compared with that of the gold standard (penicillin plus gentamicin in three divided doses) in the treatment of experimental endocarditis caused by viridans streptococci with decreased susceptibility to penicillin has not been evaluated to date. One study (6) evaluated the efficacies of netilmicin and ceftriaxone either alone or combined in the treatment of penicillin-resistant viridans streptococcal experimental endocarditis and showed successful results. In order to study all the possibilities found in the treatment of streptococcal endocarditis in humans, we used three types of strains to produce experimental endocarditis in rabbits: one with tolerance to penicillin and two with different degrees of resistance to penicillin. Results showed the combination of penicillin and gentamicin at 3 mg/kg once a day (peak concentration in serum, 8.53 g/ml) to be more effective than penicillin alone (P 0.01) and similar to the gold standard, penicillin plus gentamicin at 1 mg/kg/8 h (peak concentration in serum, 3.85 g/ml) (P 0.05), in the treatment of experimental endocarditis caused by viridans streptococci with decreased susceptibility to penicillin. Our data suggested that the increased aminoglycoside dosing interval had no influence on the in vivo efficacy of the combination of penicillin and gentamicin for the treatment of experimental endocarditis due to viridans streptococci with different susceptibilities to penicillin. The exact mechanism of this in vivo effect remains to be elucidated. Our findings could be explained by several mechanisms. The aminoglycoside bactericidal activity is concentration dependent (26). This effect is apparent in some of the time-kill curves determined in this study; in general, the higher the gentamicin concentration, the greater the bactericidal activity of the combination of penicillin Treatment group b (n) c TABLE 3. Treatment of experimental endocarditis caused by PSVS and PTVS a PSVS Log CFU/g of vegetation PTVS Mean SD Median (range) d Mean SD Median (range) d Control (6, 4) 11.3 0.48 11.38 (10.67 11.83) 11.04 0.59 11.22 (10.23 11.5) Penicillin (11, 11) 7.37 0.69 7.54 (5.79 8.36)* 8.19 1.12 8.43 (5.87 9.26)* PP G at 1 mg/kg/8 h (12, 9) 3.94 1.75 3.19 (2 6.86)* 3.79 1.78 3.29 (2 7.24)* PP G at 3 mg/kg/24 h (12, 11) 3.88 1.88 2.79 (2 6.61)* 3.02 1.5 2.43 (2 6.74)* PP G at 1 mg/kg/24 h (12) 6.24 1.65 6.73 (3.52 8.49)* ND e ND e a Both PTVS and PSVS were S. sanguis II. b PP, procaine penicillin; G, gentamicin. c The first number refers to PSVS, and the second refers to PTVS. d, P 0.01 versus control;, P 0.001 versus penicillin alone;, P 0.05 versus penicillin plus gentamicin at 1 mg/kg/8 h. e ND, not done.

2102 GAVALDÀ ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 4. Treatment of experimental endocarditis caused by PrVS and PRVS a Log CFU/g of vegetation Treatment group b (n) c PrVS PRVS Mean SD Median (range) d Mean SD Median (range) d Control (7, 5) 10.0 0.82 9.77 (8.77 11.25) 11.04 0.53 11.12 (10.12 11.46) Penicillin (15) 7.8 0.9 7.98 (5.43 9.11)* ND e ND e PP G at 1 mg/kg/8 h (15, 9) 2.33 0.5 2.0 (2 3.36)* 4.09 1.09 4.43 (2.17 5.43)* PP G at 3 mg/kg/24 h (16, 9) 3.12 1.6 2.0 (2 5.64)* 4.01 1.52 3.76 (2.6 7.13)* a The PrVS strain was S. intermedius, and the PRVS strain was S. sanguis II. b PP, procaine penicillin; G, gentamicin. c The first number refers to PrVs, and the second refers to PRVS. d, P 0.001 versus control;, P 0.0001 versus penicillin alone;, P 0.05 versus penicillin plus gentamicin at 1 mg/kg/8 h. e ND, not done. plus gentamicin. Thus, the in vivo activity of penicillin plus gentamicin once a day could be similar to that of the combination with lower doses of gentamicin administered three times a day because of the early bactericidal contribution of the aminoglycoside component, favored by the homogeneous penetration of aminoglycosides inside the vegetation, with local concentrations rapidly becoming similar to concentrations in serum (10). Likewise, our findings could be explained by a secondary effect of the -lactam after the early bactericidal effect of the combination. Bacterial regrowth inside the vegetations could be inhibited by the action of penicillin after the greater lethal action of the combination, or, furthermore, penicillin by itself could exert a bactericidal effect on streptococcal cells inside the vegetation during the period in which aminoglycoside levels are absent. Finally, the presence of an in vivo postantibiotic effect (PAE) with the combination of penicillin and gentamicin could explain, in part, our results. To our knowledge, this effect on viridans streptococci has never been studied. Such an effect was not observed in vivo in the treatment of experimental enterococcal endocarditis (22). The absence of an in vivo PAE of the combination of a -lactam plus an aminoglycoside against enterococci does not preclude the presence of a PAE against viridans streptococci because of differences in the mechanisms of action of this combination on the two microorganisms (28, 29, 44). The mechanism of synergism between penicillin and aminoglycosides against viridans streptococci is still controversial. Yee et al. (44) showed a mechanism similar to that which occurs with E. faecalis (29), but Miller et al. (28), using another strain of viridans streptococci, were unable to prove an increase in the rate of entry of aminoglycoside into bacteria in the presence of penicillin. Other mechanisms of synergy, not yet established, could explain the efficacy of gentamicin given once a day. No contradictory results regarding the minimum dose of the aminoglycoside necessary for maintaining in vivo synergism with penicillin in the treatment of penicillin-susceptible viridans streptococcal endocarditis have been published. After determining that gentamicin once a day was as effective as gentamicin three times daily, we wished to evaluate the minimum once-a-day aminoglycoside dose necessary for maintaining in vivo synergism with penicillin. Therefore, we treated a group of animals with experimental endocarditis caused by PSVS with penicillin and gentamicin at 1 mg/kg (peak concentration in serum, 3.8 g/ml), and results showed this combination to be no more effective than was penicillin alone. In the treatment of viridans streptococcal experimental endocarditis, the minimum dose of gentamicin administered once a day which maintained in vivo synergism with penicillin was 3 mg/ kg, resulting in a peak level in serum of approximately 8 g/ml. On the basis of studies discussed here (6, 16, 38) and our work, studies could be initiated to determine the combinations of procaine penicillin or other -lactams with a prolonged half-life (e.g., ceftriaxone) administered once a day and gentamicin, also given in a once-daily dose, which could be used for the treatment of nonenterococcal endocarditis in humans, with no loss of efficacy and probably with less toxicity. Hence, these regimens could make feasible the treatment of patients with nonenterococcal endocarditis at home or on an outpatient basis, and consequently they could be cost- and quality-effective alternatives. ACKNOWLEDGMENTS We thank Mark Rouse, Jim Steckelberg, and Walter Wilson, Infectious Diseases Research Laboratory, Mayo Clinic, Rochester, Minn., for valuable advice and Pere Joan Cardona and Montserrat Giménez for technical assistance. This work was supported in part by Dirección General de Investigación, Ciencia y Técnica (DGICYT grant SP89-0030) and by Comissió Interdepartamental de Recerca i Innovació Tecnològica (grant AR90-3767). REFERENCES 1. Anhalt, J. P. 1985. Antimicrobial assays, p. 691 729. In J. A. Washington II (ed.), Laboratory procedures in clinical microbiology. Springer-Verlag, New York. 2. Anhalt, J. P., and J. A. Washington II. 1985. Bactericidal tests, p. 731 745. In J. A. Washington II (ed.), Laboratory procedures in clinical microbiology. Springer-Verlag, New York. 3. Bamonte, F., S. Dionisotti, M. Gamba, E. Ongini, A. Arpini, and G. Melone. 1990. Relation of dosing regimen to aminoglycoside ototoxicity: evaluation of auditory damage in the guinea pig. Chemotherapy (Basel) 36:41 50. 4. Beaucaire, G., O. Leroy, C. Beuscart, P. Karp, C. Chidiac, and M. Caillaux. 1991. Clinical and bacteriological efficacy, and practical aspects of amikacin given once daily for severe infections. J. Antimicrob. Chemother. 27(Suppl. C):91 103. 5. Bisno, A. L., W. E. Dismukes, D. T. Durack, E. L. Kaplan, A. W. Karchmer, D. Kaye, S. H. Rahimtoola, M. A. Sande, J. P. Sanford, C. Watanakunakorn, and W. R. Wilson. 1989. Antimicrobial treatment of infective endocarditis due to viridans streptococci, enterococci, and staphylococci. JAMA 261: 1471 1477. 6. Blatter, M., U. Fluckinger, J. Entenza, M. P. Glauser, and P. Francioli. 1993. Simulated human serum profiles of one daily dose of ceftriaxone plus netilmicin in treatment of experimental streptococcal endocarditis. Antimicrob. Agents Chemother. 31:971 976. 7. Chan, G. L. C. 1989. Alternative dosing strategy for aminoglycosides: impact on efficacy, nephrotoxicity and ototoxicity. DICP Ann. Pharmacother. 23: 788 794. 8. Colton, T. 1991. Métodos no paramétricos, p. 227 236. In J. Salvat (ed.), Estadística en medicina. Salvat, Barcelona, Spain. 9. Craig, W. A., J. Redington, and S. C. Ebert. 1991. Pharmacodynamics of amikacin in vitro and in mouse thigh and lung infections. J. Antimicrob. Chemother. 27(Suppl. C):29 40. 10. Cremieux, A. C., B. Maziere, and J. M. Vallois. 1989. Evaluation of antibiotic diffusion into cardiac vegetations by quantitative autoradiography. J. Infect. Dis. 159:938 944.

VOL. 39, 1995 GENTAMICIN AND STREPTOCOCCAL EXPERIMENTAL ENDOCARDITIS 2103 11. De Broe, M. E., L. Verbist, and G. A. Verpooten. 1991. Influence of dosage schedule on renal accumulation of amikacin and tobramycin in man. J. Antimicrob. Chemother. 27(Suppl. C):41 48. 12. Durack, D. T., and P. B. Beeson. 1972. Experimental bacterial endocarditis. I. Colonisation of a sterile vegetation. Brit. J. Exp. Pathol. 53:44 49. 13. Eliopoulos, G. M., and R. C. Moellering. 1991. Laboratory methods used to assess the activity of antimicrobial combinations, p. 434 449. In V. Lorian (ed.), Antibiotics in laboratory medicine. Williams & Wilkins Co., Baltimore. 14. Fan, S. T., W. Y. Lau, C. H. Teoh-Chan, K. F. Lau, and E. H. Mauracher. 1988. Once daily administration of netilmicin compared with thrice daily, both in combination with metronidazole, in gangrenous and perforated appendicitis. J. Antimicrob. Chemother. 22:69 74. 15. Fantin, B., and C. Carbon. 1990. Importance of the aminoglycoside dosing regimen in the penicillin-netilmicin combination for treatment of Enterococcus faecalis-induced experimental endocarditis. Antimicrob. Agents Chemother. 34:2387 2391. 16. Francioli, P., and M. P. Glauser. 1993. Synergistic activity of ceftriaxone combined with netilmicin administered in one daily dose in the treatment of experimental streptococcal endocarditis. Antimicrob. Agents Chemother. 37:207 212. 17. Garrison, P., and L. R. Freedman. 1970. Experimental endocarditis. I. Staphylococcal endocarditis in rabbits resulting from placement of a polyethylene catheter in right side of the heart. Yale J. Biol. Med. 42:394 410. 17a.Gavalda, J., A. Pahissa, B. Almirante, E. Crespo, F. Fernandez, J. Martinez- Vazquez, M. Rouse, J. Steckelberg, and W. Wilson. 1992. Efficacy of gentamicin once a day in the treatment of viridans streptococcal experimental endocarditis, abstr. 1449, p. 352. In Program and abstracts of the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 18. Gerber, A. U. 1988. Comparison of once-daily versus human equivalent dosing of aminoglycoside: basic considerations and experimental approach. J. Drug Dev. 1(Suppl. 3):17 23. 19. Gerber, A. U., S. Kozak, C. Segessenmann, U. Fluckinger, T. Bangerter, and U. Greter. 1989. Once-daily versus thrice-daily administration of netilmicin in combination therapy of Pseudomonas aeruginosa in man-adapted neutropenic animal model. Eur. J. Clin. Microb. Infect. Dis. 8:233 237. 20. Giamarellou, H., K. Yiallouros, G. Petrikkos, E. Moschorabis, E. Vavouraki, D. Voutsinas, and P. Sjikakis. 1991. Comparative kinetics and efficacy of amikacin administered once or twice daily in the treatment of systemic gram-negative infections. J. Antimicrob. Chemother. 27(Suppl. C):73 79. 21. Herscovici, L., G. Grise, C. Thauvin, J. F. Lemeland, and J. P. Fillastre. 1988. Efficacy and safety of once daily versus intermittent dosing of tobramycin in rabbits with acute pyelonephritis. Scand. J. Infect. Dis. 20:205 212. 22. Hessen, M. T., P. G. Pistakis, and M. E. Levison. 1989. Postantibiotic effect of penicillin plus gentamicin versus Enterococcus faecalis in vitro and in vivo. Antimicrob. Agents Chemother. 33:608 611. 23. Hollender, L. F., J. Bahnini, N. De Manzini, W. Y. Low, S. T. Fan, K. Hermansyur, P. Benny, A. N. Husni, S. Sutzipto, and R. R. Lorber. 1989. A multicentric study of netilmicin once daily versus thrice daily in patients with appendicitis and other intra-abdominal infections. J. Antimicrob. Chemother. 23:773 783. 24. Kapusnik, J. E., C. J. Hackbarth, H. F. Chambers, T. Carpenter, and M. A. Sande. 1988. Single, large, daily dosing versus intermittent dosing of tobramycin for treating experimental Pseudomonas pneumonia. J. Infect. Dis. 158:7 12. 25. Legget, J. E., S. Ebert, B. Fantin, and W. A. Craig. 1990. Comparative dose-effect relations at several dosing intervals for beta-lactam, aminoglycoside and quinolone antibiotics against gram-negative bacilli in murine thighinfection and pneumonitis models. Scand. J. Infect. Dis. 74:179 184. 26. Legget, J. E., B. Fantin, S. Ebert, K. Totsuka, W. Calame, H. Mattie, and W. A. Craig. 1989. Comparative antibiotic dose-effect relations at several dosing intervals in murine pneumonitis and thigh-infection models. J. Infect. Dis. 159:281 292. 27. Maller, R., H. Ahrne, T. Eilard, I. Eriksson, and I. Lausen. 1991. Efficacy and safety of amikacin in systemic infections when given as a single daily dose or in two divided doses. Scandinavian Amikacin Once Daily Study Group. J. Antimicrob. Chemother. 27(Suppl. C):121 128. 28. Miller, M. H., M. A. El-Sokkary, S. A. Feinstein, and F. D. Lowy. 1986. Penicillin-induced effects on streptomycin uptake and early bactericidal activity differ in viridans group and enterococcal streptococci. Antimicrob. Agents Chemother. 30:763 768. 29. Moellering, R. C., and A. N. Weinberg. 1971. Studies on antibiotic synergism against enterococci. II. Effect of various antibiotics on the uptake of 14 C labelled streptomycin by enterococci. J. Clin. Invest. 50:2580 2584. 30. Olier, B., G. Viotte, J. P. Morin, and J. P. Fillastre. 1983. Influence of dosage regimen on experimental tobramycin nephrotoxicity. Chemotherapy (Basel) 29:385 394. 31. Pechère, M., R. Letarte, and J. C. Pechère. 1987. Efficacy of different dosing schedules of tobramycin for treating a murine Klebsiella pneumoniae bronchopneumonia. J. Antimicrob. Chemother. 19:487 491. 32. Perlman, B. B., and L. R. Freedman. 1971. Experimental endocarditis. II. Staphylococcal infection of the aortic valve following placement of a polyethylene catheter in the left side of the heart. Yale J. Biol. Med. 44:206 213. 33. Pettorossi, V. E., A. Ferraresi, P. Errico, F. Draicchio, and S. Dionisotti. 1990. The impact of different dosing regimens of the aminoglycosides netilmicin and amikacin on vestibulotoxicity in the guinea pig. Eur. Arch. Otorhinolaryngol. 247:277 282. 34. Pierre, C., F. Blanchet, N. Seta, P. Chaigne, C. Labarre, O. Sterkers, C. Amiel, and C. Carbon. 1988. Tolerance of once daily dosing of netilmicin and teicoplanin alone or in combination in healthy volunteers. Clin. Pharmacol. Ther. 44:458 466. 35. Powell, S. H., W. L. Thompson, M. A. Luthe, R. C. Stern, D. A. Grossniklaus, D. D. Blaxham, D. L. Graden, M. R. Jacobs, A. O. SiScenne, H. H. Cash, and J. D. Klinger. 1983. Once daily vs. continuous aminoglycoside dosing: efficacy and toxicity in animals and clinical studies of gentamicin, netilmicin and tobramycin. J. Infect. Dis. 147:918 932. 36. Prins, J. M., H. R. Büller, E. J. Kuijper, R. A. Tange, and P. Speelman. 1993. Once versus thrice daily gentamicin in patients with serious infections. Lancet 341:335 338. 37. Proctor, L. B., P. Petty, R. Lietman, R. Thakor, R. Glackin, and H. Shimizu. 1987. A study of potential vestibulotoxic effects of once daily versus thrice daily administrations of tobramycin. Laryngoscope 97:1443 1449. 38. Saleh-Mghir, A., A. C. Crémieux, J. M. Vallois, M. Muffat-Joly, C. Devine, and C. Carbon. 1992. Optimal aminoglycoside dosing regimen for penicillintobramycin synergism in experimental Streptococcus adjacens endocarditis. Antimicrob. Agents Chemother. 36:2043 2047. 39. Sturm, A. W. 1989. Netilmicin in the treatment of gram-negative bacteremia: single daily versus multiple daily dosage. J. Infect. Dis. 159:931 937. 40. ter Braak, E. W., P. J. de Vries, K. P. Bouter, S. G. van der Vegt, G. C. Dorrestein, J. W. Nortier, A. van Dijk, R. P. Verkooyen, and H. A. Verbrugh. 1990. Once-daily dosing regimen for aminoglycoside plus beta-lactam combination therapy of serious bacterial infections: comparative trial with netilmicin plus ceftriaxone. Am. J. Med. 89:58 66. 41. Tulkens, P. M. 1991. Pharmacokinetic and toxicological evaluation of a once-daily regimen versus conventional schedules of netilmicin and amikacin. J. Antimicrob. Chemother. 27(Suppl. C):49 61. 42. Verpooten, G. A., R. A. Giulano, L. Verbist, G. Eestermans, and M. E. De Broe. 1989. Once daily dosing decreases renal accumulation of gentamicin and netilmicin. Clin. Pharmacol. Ther. 45:22 27. 43. Wood, C. A., D. R. Norton, S. J. Kohlhepp, P. W. Kohnen, G. A. Porter, D. C. Houghton, R. E. Brummett, W. M. Bennett, and D. N. Gilbert. 1988. The influence of tobramycin dosage regimen on nephrotoxicity, ototoxicity and antibacterial efficacy in a rat model of subcutaneous abscess. J. Infect. Dis. 158:13 22. 44. Yee, Y., B. Farber, and S. Mates. 1986. Mechanism of penicillin-streptomycin synergy for clinical isolates of viridans streptococci. J. Infect. Dis. 154:531 534.