ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1992, p. 1256-1262
Vol. 36, No. 6
0066-4804/92/061256-07$02.00/0 Copyright © 1992, American Society for Microbiology
Teicoplanin versus Vancomycin for Prophylaxis of Experimental Enterococcus faecalis Endocarditis in Rats JOSE M. ENTENZA, THIERRY CALANDRA, YVES MOOSMANN,t RAFFAELE MALINVERNI,4 AND MICHEL P. GLAUSER* Division of Infectious Diseases, Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Received 18 December 1991/Accepted 6 April 1992
Teicoplanin was compared with vancomycin for the prophylaxis of experimental Enterococcus faecalis endocarditis in rats. Single intravenous doses of teicoplanin (7 mg/kg of body weight) or vancomycin (15 mg/kg) were given 30 min before bacterial challenge. Two strains of E. faecalis (309 and 1209) isolated from patients with endocarditis were tested. Bacterial inocula ranged from 104 (i.e., the inoculum infecting 90%o of the control rats [ID901) to 107 CFU/ml. The MICs and MBCs of teicoplanin and vancomycin were, respectively, 0.25 to > 128 mg/liter and 2 to > 128 mg/liter for strain 309 and 0.5 to > 128 mg/liter and 0.5 to > 128 mg/liter for strain 1209. Vancomycin prevented endocarditis only in 60% (strain 309) and in 87% (strain 1209) of rats challenged with the smallest bacterial-inoculum size (ID0), whereas teicoplanin prevented endocarditis in 100% of rats challenged with the same inoculum (strain 309; P = 0.05), in 87% of rats challenged with 10 times the ID90 (strain 309; P = 0.02), and in 95% of rats challenged with 100 times the ID90 (strain 1209; P = 0.0003). The combination of teicoplanin plus gentamicift (4 mg/kg) extended the protection to inocula 100 times the ID90 (strain 309; 96% of sterile animals) and 1,000 times the ID90 (strain 1209; 100%o of sterile animals). Prevention of endocarditis was likely to be due to a prolonged inhibition of bacterial growth by sustained levels of teicoplanin in serum and not to bacterial killing. Indeed, teicoplanin did not exhibit any bactericidal activity either in vitro (time-kill curves) or in vivo (serum bactericidal activity). Teicoplanin proved to be superior to vancomycin in the prophylaxis of experimental E. faecalis endocarditis in rats.
Teicoplanin is a glycopeptide antibiotic chemically related to vancomycin (20). These two antibiotics have similar
MATERIALS AND METHODS Microorganisms. We used two strains of E. faecalis (309 and 1209) originally isolated from patients with endocarditis. These two E. faecalis strains have been previously evaluated in the same endocarditis model in rats (5, 14). The bacteria were stored at -80°C in horse blood and subcultured on 5% human blood agar plates 1 to 5 days before each experiment. Determination of MICs, MBCs, and rates of killing. The MICs of teicoplanin (Merrel Dow, Zurich, Switzerland), of vancomycin (Eli Lilly, Indianapolis, Ind.), and of gentamicin (Essex Chemie, Luzern, Switzerland) for the two E. faecalis strains were determined by using a standard broth dilution technique, with an inoculum of 106 organisms per ml from an overnight culture (11). The MIC was determined as the lowest antibiotic concentration preventing visible growth. The MBCs were determined by subculturing onto blood agar plates 10- and 100-fold dilutions of a 0.1-ml sample from each dilution of antibiotic showing no turbidity after 18 h of incubation. This dilution procedure ensured that the carryover of antibiotics did not inhibit colony formation onto plates. After a 48-h incubation of the blood agar plates, the number of colonies from each subculture was counted, and the MBC was determined as the lowest dilution of antibiotic that showed 99.9% killing of the initial inoculum. Killing curves were determined in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) with an inoculum of 106 CFU/ml from an overnight culture by using concentrations of 32 mg of teicoplanin per liter (corresponding to 128 and 64 times the MICs for strains 309 and 1209, respectively), of 36 mg of vancomycin per liter (corresponding to 18 and 72 times the MICs for strains 309 and 1209, respectively), and of 8 mg of gentamicin per liter (corresponding to the MIC for strain 309 and to twice the MIC for strain 1209). These concentrations of teicoplanin, vancomycin, and gentamicin were cho-
modes of action (26) and comparable in vitro activities against a wide spectrum of gram-positive bacteria, including enterococci and methicillin-susceptible and methicillin-resistant staphylococci (8, 18, 19). Unlike vancomycin, however, teicoplanin displays an extremely long half-life in plasma in vivo, allowing once-daily administration, and is well tolerated when given intramuscularly (28, 29). In view of its spectrum of antibacterial activity and its pharmacokinetic properties, teicoplanin may be an alternative to vancomycin. Enterococcal endocarditis is the third most common cause of infective endocarditis (12) and is difficult to treat because of an increased resistance to P-lactam antibiotics and aminoglycosides (10). In patients allergic to P-lactam antibiotics, vancomycin is the recommended antibiotic for the prophylaxis of bacterial endocarditis, especially in patients undergoing gastrointestinal or genitourinary procedures (4, 23, 24). The aim of the present study was to investigate the efficacy of teicoplanin for the prophylaxis of experimental Enterococcus faecalis endocarditis in rats and to compare it with that of vancomycin. (This work was presented in part at the 27th Interscience Conference on Antimicrobial Agents and Chemotherapy [4a], October 1987, New York, N.Y.)
*
Corresponding author.
t Present address: Institut de Zoologie, Universite de Neuchatel, CH-2007 Neuchatel, Switzerland. t Present address: Medizinische Poliklinik, Inselspital, CH-3010 Bern, Switzerland. 1256
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sen because they were similar to the peak levels in serum obtained in rats 30 min after the intravenous injection of 7 mg of teicoplanin per kg of body weight, 15 mg of vancomycin per kg, and 4 mg of gentamicin per kg. At various times after inoculation of the bacteria into the antibiotic-containing broth, 10-1, 10-3, and 10-5 dilutions of a 0.1-ml sample were subcultured onto blood agar plates and were incubated for 48 h for colony counts. Drugs kinetics. Concentrations of antibiotics in serum were determined by a standard agar diffusion technique (21) 30 min and 1, 2, 4, 6, 8, 12, and 24 h after the intravenous injection of 7 mg of teicoplanin per kg, 15 mg of vancomycin per kg, and 4 mg of gentamicin per kg. For each time point, groups of three rats were used to determine the levels of antibiotics in serum. Bacillus subtilis (Subtilis spore suspension; Difco Laboratories) was used as the test organism, and normal rat serum was used as the diluent. The area under the mean concentration (in serum)-time curve (AUC) and the AUC above the MICs of teicoplanin and vancomycin were calculated by the log trapezoidal method with extrapolation to infinity (6). Determination of the serum bactericidal activity. The serum bactericidal activity 30 min and 1, 2, and 4 h after the intravenous administration of teicoplanin and vancomycin to rats was determined for each strain by standard methods using an inoculum of 106 CFU/ml from an overnight culture (22). Subcultures were performed on blood agar by plating 10- and 100-fold dilutions of a 0.1-ml sample of each dilution. The serum bactericidal activity was defined as the highest serum dilution providing 99.9% killing of the initial inoculum after 18 h of incubation. Production of endocarditis and evaluation of infection. Sterile vegetations were produced in female Wistar rats (180 to 200 g) by a modification of a previously described method (9). In brief, a polyethylene catheter (PP 10; Portex, Hythe, Kent, England) was inserted, via the right carotid artery, through the aortic valve into the left ventricle and secured with a silk ligature. Twenty-four hours after catheterization, rats were injected in the tail vein with 0.5 ml of saline containing various inocula from an overnight culture of each E. faecalis strain tested. The number of bacteria injected intravenously was confirmed by colony counts and expressed in CFU per milliliter. The lowest bacterial inoculum infecting 90% of the control rats (ID90) was 104 CFU/ml for both E. faecalis strains. Rats were killed 72 h after singledose prophylaxis. The aortic vegetations were excised, weighed, homogenized in 1 ml of saline, serially diluted, and plated onto blood agar. The colonies were counted after 48 h of incubation at 37°C. This method permitted the detection of 102 CFU/g of vegetation. Prophylaxis of endocarditis with teicoplanin, vancomycin, and teicoplanin plus gentamicin. Thirty minutes before bacterial challenge, rats were randomly divided into three groups. The first group received a single intravenous dose of 7 mg of teicoplanin per kg. The second group received a single intravenous dose of 15 mg of vancomycin per kg. The third group (controls) received 0.5 ml of normal saline intravenously. For each E. faecalis strain, two additional groups of rats infected with high bacterial inocula (i.e., 106 and 107 CFU/ml, corresponding to 100 and 1,000 times the ID90) received prophylaxis consisting of the combination of a single intravenous dose of 7 mg of teicoplanin per kg and 4 mg of gentamicin per kg. These concentrations of teicoplanin, vancomycin, and gentamicin were chosen because they produced peak levels in serum similar to those found in
1257
TABLE 1. MICs and MBCs of teicoplanin, vancomycin, and gentamicin for the two E. faecalis strains tested Concn (mg/liter) of:
Strain
309 1209
Teicoplanin
Vancomycin
Gentamicin
MIC
MBC
MIC
MBC
MIC
MBC
0.25 0.5
> 128 >128
2 0.5
> 128 >128
8 4
64 16
humans after the administration of therapeutic doses of these antibiotics (16, 25, 27). Statistical evaluation. The chi-square test with Yates' correction and the unpaired Student's t test were used for statistical comparisons. All reported significance levels are two sided. The Bonferroni method was used to adjust P values for multiple comparisons (1).
RESULTS MICs and MBCs. The MICs and MBCs of teicoplanin, vancomycin, and gentamicin for the two E. faecalis strains tested are shown in Table 1. For strain 309 the MIC of teicoplanin was lower than that of vancomycin, whereas they were equal for strain 1209. Both strains had high MBCs of teicoplanin and vancomycin, which is a common phenomenon for E. faecalis when cell wall-active antibiotics are tested (15). Drugs kinetics. Peak concentrations of teicoplanin, vancomycin, and gentamicin in serum, measured 30 min after the intravenous bolus injection of the antibiotic (i.e., at the time of bacterial challenge), were 32, 36, and 8 mg/liter, respectively. The concentrations of gentamicin and vancomycin in serum were undetectable 4 and 6 h, respectively, after the injection of antibiotic, whereas low concentrations of teicoplanin in serum were still measurable after 18 h (Fig. 1). The AUCs of teicoplanin and vancomycin were, respectively, 208 and 61 mg h/liter, and the AUCs above the MIC were 199 and 52 mg h/liter for strain 309 and 191 and 58 mg. h/ liter for strain 1209. Serum bactericidal activities and rates of killing. Following the intravenous injection of 7 mg of teicoplanin per kg or 15 mg of vancomycin per kg, no bactericidal activity could be detected at any time in the serum of rats. Figure 2 shows the rates of in vitro killing of the two E. faecalis strains tested by 32 mg of teicoplanin per liter, 36 mg of vancomycin per liter, 8 mg of gentamicin per liter, and the combination of teicoplanin and gentamicin. Neither of the E. faecalis strains showed a reduction of the bacterial counts within 48 h of exposure to peak teicoplanin or vancomycin concentrations. When exposed to gentamicin, both strains showed a 2 log1o decrease of the colony counts over 24 h. However, regrowth of the bacteria occurred at 48 h and was more pronounced for strain 309 than for strain 1209. In contrast, when exposed to a combination of teicoplanin and gentamicin, both E. faecalis strains were killed within 24 h of incubation. Prophylaxis with teicoplanin, vancomycin, and teicoplanin and gentamicin. The results of prophylaxis against the two E. faecalis strains tested are shown in Fig. 3. The ID90 was 104 CFU/ml for each E. faecalis strain. In rats challenged with this inoculum teicoplanin completely prevented endocarditis (strain 309, no rats infected; P < 10-5 versus controls). Vancomycin significantly reduced the incidence of infection
1258
ANTIMICROB. AGENTS CHEMOTHER.
ENTENZA ET AL.
7mg/kg
E 4)
c)
.51 2
4
24 Hours
12
FIG. 1. Concentrations of teicoplanin, vancomycin, and gentamicin in serum in rats after a single intravenous bolus injection.
(P < 0.05 compared with controls), but prophylactic failures occurred in 8 of 20 rats (40%) challenged with E. faecalis 309 (P = 0.05 versus teicoplanin). With inocula 10 times higher than the ID90 (i.e., 105 CFU/ml), prophylaxis with teicoplanin was successful against both strains of E. faecalis (strain 309, 2 rats (13%) infected; P < 10-5; strain 1209, no rats infected; P < 10-4). Prophylaxis with vancomycin failed for both strains; 64% of the rats were infected with strain 309 (P = 0.02 versus teicoplanin), and 80% were infected with strain 1209 (P = 0.0005 versus teicoplanin). With inocula 100 times higher than the ID90 (i.e., 106 CFU/ml) vancomycin completely failed to prevent endocarditis. At this inoculum, teicoplanin almost completely protected the animals challenged with E. faecalis 1209 (1 rat of 18 was infected; P < 10-4 compared with controls; P = 0.0003 versus vancomycin). Teicoplanin also significantly prevented endocarditis in rats challenged with E. faecalis 309, but the failure rate was 37% (6 rats of 16 were infected; P = 0.001 versus controls; P = 0.008 versus vancomycin). With greater inocula (1,000 times the IDO, i.e., 107 CFU/ ml) the prophylactic effect of teicoplanin was overcome. The combination of teicoplanin plus gentamicin extended the protection conferred by teicoplanin alone to inocula 100 times the ID90 (i.e., 106 CFU/ml) in rats challenged with E. faecalis 309 (infected rats: 4% for teicoplanin plus gentamicin versus 37% for teicoplanin; P = 0.08) and to inocula 1,000 times the ID90 (i.e., 107 CFU/ml) in rats challenged with E. faecalis 1209 (infected rats: 0% for teicoplanin plus gentamicin versus 86% for teicoplanin alone; P < 10-5). Of note, for all prophylactic groups, the mean bacterial densities of the vegetations of the rats in which teicoplanin prophylaxis failed were significantly inferior to those of controls (P < 0.005). In contrast, the mean bacterial densities of the vegetations of the rats in which vancomycin prophylaxis failed were similar to those of controls.
DISCUSSION This study compared the efficacy of a single dose of teicoplanin with that of vancomycin for the prophylaxis of experimental enterococcal endocarditis in rats. For the two E. faecalis strains tested, vancomycin was only partially successful against an inoculum equal to the ID90, while teicoplanin prevented endocarditis against inocula 10 to 100 times higher than the ID90. This protection was conferred in the absence of bacterial killing. Indeed, no bactericidal activity was detected at any time in the serum of rats, and the time-kill curves did not show a significant reduction in bacterial numbers at peak concentrations of teicoplanin and vancomycin in serum. Previous studies in rats have shown that when viridans group streptococci or enterococci either were not rapidly killed or were tolerant to amoxicillin, single-dose prophylaxis of endocarditis was efficacious only at the ID90 (5). Against inocula higher than the ID90, only multiple doses of amoxicillin (viridans group streptococci) or amoxicillin plus gentamicin (enterococci) were necessary for successful prophylaxis (14). In the absence of bacterial killing, early studies (2, 7) have suggested that the inhibition of bacterial adherence to vegetations was the mechanism by which prophylaxis prevented endocarditis. However, recent observations (13, 17) have shown that prolonged inhibition of bacterial growth after the intravenous challenge, rather than inhibition of bacterial adherence, was likely to be the mechanism of protection. The longer the duration of growth inhibition is, the greater the likelihood of successful prophylaxis after challenge with inocula exceeding the ID90 is. These results suggested that prophylactic antibiotics allowed the adherent bacteria to be cleared from the valves by an as yet undetermined mechanism. Single doses of antibiotics, even when used in combination (5), have failed to protect against inocula higher the IDg, probably because blood antibiotic levels were not sustained for long enough to
TEICOPLANIN PROPHYLAXIS FOR ENTEROCOCCAL ENDOCARDITIS
VOL. 36, 1992
1259
E. faecalis 309
E-I L
0
0 2 4 6
12
24
48
Hours of incubation
E. faecalis 1209
UL
0) 0
0 2 4 6
12
24
48
Hours of incubation FIG. 2. Killing of the two E. faecalis strains tested by 32 mg of teicoplanin per liter, 36 mg of vancomycin per liter, 8 mg of gentamicin per liter, and the combination of teicoplanin and gentamicin. The antibiotic concentrations were similar to peak levels in serum obtained in rats 30 min after the intravenous injection of 7 mg of teicoplanin per kg, 15 mg of vancomycin per kg, and 4 mg of gentamicin per kg.
1260
ANTIMICROB. AGENTS CHEMOTHER.
ENTENZA ET AL.
E. faecalis 309 *
Log 10 MBD
7;9
:t SD
3
8.38 8.37
i0.52
-
0.69
-
*
&64 8.19 5.04
10.66
0.91
0.541
*
s85 8.96 6.25 4.24 0.40 0.60
1.07
-
9.00
6.25 4.72
103
090
0651
11 U) 0
0 -W
0 0
0 :m 0
*_
0 oc
1-
Inoculum: 103
104
1o-05
167
106
E. faecalis 1209 *
Log 10 MBD & SD
U) c
i [.
7 1.2
8
8.82 7.11
8.4
-
9
OA
L.9
8u5.56 -
5.26
1-4
3
113
1.7
.
1
0
4-.
4-0
0)
10 0-
0
I.-
*
0
22
12
Inoculum
106 105 1003
167
I
TEICOPLANIN PROPHYLAXIS FOR ENTEROCOCCAL ENDOCARDITIS
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1261
FIG. 3. Incidence of endocarditis in control rats (C) and in rats given single prophylactic dose of teicoplanin (T), vancomycin (V), or teicoplanin plus gentamicin (T+G), 30 min before challenge with various inocula of the two E. faecalis strains. The total number of rats is indicated at the base of each column. The mean bacterial densities (MBD) cultured from vegetations (expressed as log,( CFU per gram of vegetation) is indicated for each group. Statistical comparisons of prophylactic groups with controls: *, P < 0.05; t, P < 0.00001.
completely eliminate the bacteria attached to the vegetations. In the present study, a single intravenous dose of teicoplanin produced levels in serum which were above the MICs for the two E. faecalis strains tested for more than 12 h after the intravenous injection. In contrast, levels of vancomycin in serum were above the MIC for no longer than 4 h. Thus, it is likely that the better prophylactic effect of teicoplanin was due to a longer inhibition of bacterial growth, as shown by a greater AUC above the MIC compared with that of vancomycin (-190 versus -50 mg. h/liter). The finding that bacterial densities in vegetations of rats in which prophylaxis failed were lower in the teicoplanin groups than in either the vancomycin or the control groups supports this hypothesis. The fact that teicoplanin is retained at the periphery of the vegetations and fails to diffuse into their core (3) may contribute to its failure in the treatment of endocarditis, but as suggested by our results, this phenomenon does not impair its prophylactic efficacy, probably because the bacteria are at the surface of the vegetations not yet protected by fibrin. In conclusion, teicoplanin was superior to vancomycin for the prevention of E. faecalis endocarditis in rats. ACKNOWLEDGMENTS We thank M. Giddey and M. Knaup for technical work, A. Munafo for assistance with AUC analysis, and M. M. Wu for statistical consultation.
REFERENCES 1. Altman,D. G. 1991. Practical statistics for medical research, p. 210. Chapman and Hall, New York. 2. Bernard, J. P., P. Francioli, and M. P. Glauser. 1981. Vancomycin prophylaxis of streptococcal endocarditis. Inhibition of bacterial adherence rather than bacterial killing. J. Clin. Invest. 68:1113-1116. 3. Cremieux, A. C., B. Maziere, J. M. Vallois, M. Ottaviani, A. Azancot, H. Raffoul, A. Bouvet, J. J. Pocidalo, and C. Carbon. 1989. Evaluation of antibiotic diffusion into cardiac vegetations by quantitative autoradiography. J. Infect. Dis. 159:938-944. 4. Endocarditis Working Party. 1990. Antibiotic prophylaxis of infective endocarditis. Recommendations from the Endocarditis Working Party of the British Society for Antimicrobial Chemotherapy. Lancet 335:88-89. 4a.Entenza, J., T. Calandra, and M. P. Glauser. 1987. Program Abstr. 27th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 928. 5. Francioli, P., P. Moreillon, and M. P. Glauser. 1985. Comparison of single doses of amoxicillin or of amoxicillin-gentamicin for the prevention of endocarditis caused by Streptococcus faecalis and by viridans streptococci. J. Infect. Dis. 152:83-89. 6. Gibaldi, M., and D. Perrier. 1982. Pharmacokinetics, 2nd ed., p. 449. Marcel Dekker, Inc., New York. 7. Glauser, M. P., J. P. Bernard, and P. Moreillon. 1983. Successful single-dose amoxicillin prophylaxis against experimental endocarditis: evidence of two mechanisms of protection. J. Infect. Dis. 147:568-575. 8. Gorzynski, E. A., D. Amsterdam, T. R. Beam, Jr., and C. Rotstein. 1989. Comparative in vitro activities of teicoplanin, vancomycin, oxacillin, and other antimicrobial agents against bacteremic isolates of gram-positive cocci. Antimicrob. Agents Chemother. 33:2019-2022.
9. Heraief, E., M.-P. Glauser, and L. R. Freedman. 1980. Vancomycin prophylaxis of streptococcal endocarditis in rats, p. 911-913. In J. D. Nelson and C. Grassi (ed.), Current chemotherapy and infectious disease, vol. II. American Society for Microbiology, Washington, D.C. 10. Hindes, R. G., S. H. Willey, G. M. Eliopoulos, L. B. Rice, C. T. Eliopoulos, B. E. Murray, and R. C. Moellering. 1989. Treatment of experimental endocarditis caused by a 1-lactamaseproducing strain of Enterococcus faecalis with high-level resistance to gentamicin. Antimicrob. Agents Chemother. 33:10191022. 11. Jones, R. N., A. L. Barry, T. L. Gavan, and J. A. Washington II. 1985. Susceptibility tests: microdilution and macrodilution broth procedures, p. 972-977. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 12. Kaye, D. 1976. Infective microorganisms, p. 43-54. In D. Kaye (ed.), Infective endocarditis. University Park Press, Baltimore. 13. Malinverni, R., J. Bille, and M. P. Glauser. 1987. Single-dose rifampin prophylaxis for experimental endocarditis induced by high bacterial inocula of viridans streptococci. J. Infect. Dis. 156:151-157. 14. Malinverni, R., P. Francioli, and M. P. Glauser. 1987. Comparison of single and multiple doses of prophylactic antibiotics in experimental streptococcal endocarditis. Circulation 76:376382. 15. Moellering, R. C., Jr. 1981. Antimicrobial susceptibility of enterococci: in vitro studies of the action of antibiotics alone and in combination, p. 81-96. In A. L. Bisno (ed.), Treatment of infective endocarditis. Grune and Stratton, Inc., New York. 16. Moellering, R. C., Jr. 1984. Pharmacokinetics of vancomycin. J. Antimicrob. Chemother. 14(Suppl. D):43-52. 17. Moreillon, P., P. Francioli, D. Overholzer, P. Meylan, and M. P. Glauser. 1986. Mechanisms of successful amoxicillin prophylaxis of experimental endocarditis. J. Infect. Dis. 154:801-807. 18. Neu, H., and P. Labthavikul. 1983. In vitro study of theicomycin compared with those of other antibiotics. Antimicrob. Agents Chemother. 24:425-428. 19. Pallanza, R., M. Berti, B. P. Goldstein, E. Mapelli, E. Randesi, R. Scotti, and V. Airoli. 1983. Theicomycin: in vitro and in vivo evaluation in comparison with other antibiotics. J. Antimicrob. Chemother. 11:419-425. 20. Parenti, F., G. Beretta, M. Berti, and V. Airoli. 1978. Teichomycins: new antibiotics from Actinoplanes teichomyceticus nov. sp. 1. Description of the producer strain, fermentation studies and biological properties. J. Antibiot. 1:276-283. 21. Sabath, L. D., and J. P. Anhalt. 1980. Assay of antibiotics, p. 485-490. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manual of clinical Microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. 22. Schoenknecht, F. D., L. D. Sabath, and C. Thornsberry. 1985. Susceptibility tests: special tests, p. 1000-1008. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 23. Schweizerische Arbeitsgruppe fiur Endokarditisprophylaxe. 1984. Prophylaxe der backteriellen Endokarditis. Schweiz. Med. Wochenschr. 114:1246-1252. 24. Shulman, S. T., D. P. Amren, A. L. Bisno, A. S. Dajani, D. T. Durack, M. A. Gerber, E. L. Kaplan, H. D. Millard, W. E. Sanders, R. H. Schwartz, and C. Watanakunakorn. 1984. Prevention of bacterial endocarditis: a statement for health professionals by the Committee on Rheumatic Fever and Infective Endocarditis of the Council on Cardiovascular Disease in the Young. Circulation 70:1123A-1127A.
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25. Siber, G. R., P. Echeverria, A. L. Smith, J. W. Paisley, and D. H. Smith. 1975. Pharmacokinetics of gentamicin in children and adults. J. Infect. Dis. 132:637-651. 26. Somma, S., L. Gastaldo, and A. Corti. 1984. Teicoplanin, a new antibiotic from Actinoplanes teichomyceticus nov. sp. Antimicrob. Agents Chemother. 26:917-923. 27. Stille, W., W. Sietzen, H. A. Dieterich, and J. J. Fell. 1988. Clinical efficacy and safety of teicoplanin. J. Antimicrob. Che-
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0066-4804/92/061256-07$02.00/0 Copyright © 1992, American Society for Microbiology
Teicoplanin versus Vancomycin for Prophylaxis of Experimental Enterococcus faecalis Endocarditis in Rats JOSE M. ENTENZA, THIERRY CALANDRA, YVES MOOSMANN,t RAFFAELE MALINVERNI,4 AND MICHEL P. GLAUSER* Division of Infectious Diseases, Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Received 18 December 1991/Accepted 6 April 1992
Teicoplanin was compared with vancomycin for the prophylaxis of experimental Enterococcus faecalis endocarditis in rats. Single intravenous doses of teicoplanin (7 mg/kg of body weight) or vancomycin (15 mg/kg) were given 30 min before bacterial challenge. Two strains of E. faecalis (309 and 1209) isolated from patients with endocarditis were tested. Bacterial inocula ranged from 104 (i.e., the inoculum infecting 90%o of the control rats [ID901) to 107 CFU/ml. The MICs and MBCs of teicoplanin and vancomycin were, respectively, 0.25 to > 128 mg/liter and 2 to > 128 mg/liter for strain 309 and 0.5 to > 128 mg/liter and 0.5 to > 128 mg/liter for strain 1209. Vancomycin prevented endocarditis only in 60% (strain 309) and in 87% (strain 1209) of rats challenged with the smallest bacterial-inoculum size (ID0), whereas teicoplanin prevented endocarditis in 100% of rats challenged with the same inoculum (strain 309; P = 0.05), in 87% of rats challenged with 10 times the ID90 (strain 309; P = 0.02), and in 95% of rats challenged with 100 times the ID90 (strain 1209; P = 0.0003). The combination of teicoplanin plus gentamicift (4 mg/kg) extended the protection to inocula 100 times the ID90 (strain 309; 96% of sterile animals) and 1,000 times the ID90 (strain 1209; 100%o of sterile animals). Prevention of endocarditis was likely to be due to a prolonged inhibition of bacterial growth by sustained levels of teicoplanin in serum and not to bacterial killing. Indeed, teicoplanin did not exhibit any bactericidal activity either in vitro (time-kill curves) or in vivo (serum bactericidal activity). Teicoplanin proved to be superior to vancomycin in the prophylaxis of experimental E. faecalis endocarditis in rats.
Teicoplanin is a glycopeptide antibiotic chemically related to vancomycin (20). These two antibiotics have similar
MATERIALS AND METHODS Microorganisms. We used two strains of E. faecalis (309 and 1209) originally isolated from patients with endocarditis. These two E. faecalis strains have been previously evaluated in the same endocarditis model in rats (5, 14). The bacteria were stored at -80°C in horse blood and subcultured on 5% human blood agar plates 1 to 5 days before each experiment. Determination of MICs, MBCs, and rates of killing. The MICs of teicoplanin (Merrel Dow, Zurich, Switzerland), of vancomycin (Eli Lilly, Indianapolis, Ind.), and of gentamicin (Essex Chemie, Luzern, Switzerland) for the two E. faecalis strains were determined by using a standard broth dilution technique, with an inoculum of 106 organisms per ml from an overnight culture (11). The MIC was determined as the lowest antibiotic concentration preventing visible growth. The MBCs were determined by subculturing onto blood agar plates 10- and 100-fold dilutions of a 0.1-ml sample from each dilution of antibiotic showing no turbidity after 18 h of incubation. This dilution procedure ensured that the carryover of antibiotics did not inhibit colony formation onto plates. After a 48-h incubation of the blood agar plates, the number of colonies from each subculture was counted, and the MBC was determined as the lowest dilution of antibiotic that showed 99.9% killing of the initial inoculum. Killing curves were determined in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) with an inoculum of 106 CFU/ml from an overnight culture by using concentrations of 32 mg of teicoplanin per liter (corresponding to 128 and 64 times the MICs for strains 309 and 1209, respectively), of 36 mg of vancomycin per liter (corresponding to 18 and 72 times the MICs for strains 309 and 1209, respectively), and of 8 mg of gentamicin per liter (corresponding to the MIC for strain 309 and to twice the MIC for strain 1209). These concentrations of teicoplanin, vancomycin, and gentamicin were cho-
modes of action (26) and comparable in vitro activities against a wide spectrum of gram-positive bacteria, including enterococci and methicillin-susceptible and methicillin-resistant staphylococci (8, 18, 19). Unlike vancomycin, however, teicoplanin displays an extremely long half-life in plasma in vivo, allowing once-daily administration, and is well tolerated when given intramuscularly (28, 29). In view of its spectrum of antibacterial activity and its pharmacokinetic properties, teicoplanin may be an alternative to vancomycin. Enterococcal endocarditis is the third most common cause of infective endocarditis (12) and is difficult to treat because of an increased resistance to P-lactam antibiotics and aminoglycosides (10). In patients allergic to P-lactam antibiotics, vancomycin is the recommended antibiotic for the prophylaxis of bacterial endocarditis, especially in patients undergoing gastrointestinal or genitourinary procedures (4, 23, 24). The aim of the present study was to investigate the efficacy of teicoplanin for the prophylaxis of experimental Enterococcus faecalis endocarditis in rats and to compare it with that of vancomycin. (This work was presented in part at the 27th Interscience Conference on Antimicrobial Agents and Chemotherapy [4a], October 1987, New York, N.Y.)
*
Corresponding author.
t Present address: Institut de Zoologie, Universite de Neuchatel, CH-2007 Neuchatel, Switzerland. t Present address: Medizinische Poliklinik, Inselspital, CH-3010 Bern, Switzerland. 1256
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sen because they were similar to the peak levels in serum obtained in rats 30 min after the intravenous injection of 7 mg of teicoplanin per kg of body weight, 15 mg of vancomycin per kg, and 4 mg of gentamicin per kg. At various times after inoculation of the bacteria into the antibiotic-containing broth, 10-1, 10-3, and 10-5 dilutions of a 0.1-ml sample were subcultured onto blood agar plates and were incubated for 48 h for colony counts. Drugs kinetics. Concentrations of antibiotics in serum were determined by a standard agar diffusion technique (21) 30 min and 1, 2, 4, 6, 8, 12, and 24 h after the intravenous injection of 7 mg of teicoplanin per kg, 15 mg of vancomycin per kg, and 4 mg of gentamicin per kg. For each time point, groups of three rats were used to determine the levels of antibiotics in serum. Bacillus subtilis (Subtilis spore suspension; Difco Laboratories) was used as the test organism, and normal rat serum was used as the diluent. The area under the mean concentration (in serum)-time curve (AUC) and the AUC above the MICs of teicoplanin and vancomycin were calculated by the log trapezoidal method with extrapolation to infinity (6). Determination of the serum bactericidal activity. The serum bactericidal activity 30 min and 1, 2, and 4 h after the intravenous administration of teicoplanin and vancomycin to rats was determined for each strain by standard methods using an inoculum of 106 CFU/ml from an overnight culture (22). Subcultures were performed on blood agar by plating 10- and 100-fold dilutions of a 0.1-ml sample of each dilution. The serum bactericidal activity was defined as the highest serum dilution providing 99.9% killing of the initial inoculum after 18 h of incubation. Production of endocarditis and evaluation of infection. Sterile vegetations were produced in female Wistar rats (180 to 200 g) by a modification of a previously described method (9). In brief, a polyethylene catheter (PP 10; Portex, Hythe, Kent, England) was inserted, via the right carotid artery, through the aortic valve into the left ventricle and secured with a silk ligature. Twenty-four hours after catheterization, rats were injected in the tail vein with 0.5 ml of saline containing various inocula from an overnight culture of each E. faecalis strain tested. The number of bacteria injected intravenously was confirmed by colony counts and expressed in CFU per milliliter. The lowest bacterial inoculum infecting 90% of the control rats (ID90) was 104 CFU/ml for both E. faecalis strains. Rats were killed 72 h after singledose prophylaxis. The aortic vegetations were excised, weighed, homogenized in 1 ml of saline, serially diluted, and plated onto blood agar. The colonies were counted after 48 h of incubation at 37°C. This method permitted the detection of 102 CFU/g of vegetation. Prophylaxis of endocarditis with teicoplanin, vancomycin, and teicoplanin plus gentamicin. Thirty minutes before bacterial challenge, rats were randomly divided into three groups. The first group received a single intravenous dose of 7 mg of teicoplanin per kg. The second group received a single intravenous dose of 15 mg of vancomycin per kg. The third group (controls) received 0.5 ml of normal saline intravenously. For each E. faecalis strain, two additional groups of rats infected with high bacterial inocula (i.e., 106 and 107 CFU/ml, corresponding to 100 and 1,000 times the ID90) received prophylaxis consisting of the combination of a single intravenous dose of 7 mg of teicoplanin per kg and 4 mg of gentamicin per kg. These concentrations of teicoplanin, vancomycin, and gentamicin were chosen because they produced peak levels in serum similar to those found in
1257
TABLE 1. MICs and MBCs of teicoplanin, vancomycin, and gentamicin for the two E. faecalis strains tested Concn (mg/liter) of:
Strain
309 1209
Teicoplanin
Vancomycin
Gentamicin
MIC
MBC
MIC
MBC
MIC
MBC
0.25 0.5
> 128 >128
2 0.5
> 128 >128
8 4
64 16
humans after the administration of therapeutic doses of these antibiotics (16, 25, 27). Statistical evaluation. The chi-square test with Yates' correction and the unpaired Student's t test were used for statistical comparisons. All reported significance levels are two sided. The Bonferroni method was used to adjust P values for multiple comparisons (1).
RESULTS MICs and MBCs. The MICs and MBCs of teicoplanin, vancomycin, and gentamicin for the two E. faecalis strains tested are shown in Table 1. For strain 309 the MIC of teicoplanin was lower than that of vancomycin, whereas they were equal for strain 1209. Both strains had high MBCs of teicoplanin and vancomycin, which is a common phenomenon for E. faecalis when cell wall-active antibiotics are tested (15). Drugs kinetics. Peak concentrations of teicoplanin, vancomycin, and gentamicin in serum, measured 30 min after the intravenous bolus injection of the antibiotic (i.e., at the time of bacterial challenge), were 32, 36, and 8 mg/liter, respectively. The concentrations of gentamicin and vancomycin in serum were undetectable 4 and 6 h, respectively, after the injection of antibiotic, whereas low concentrations of teicoplanin in serum were still measurable after 18 h (Fig. 1). The AUCs of teicoplanin and vancomycin were, respectively, 208 and 61 mg h/liter, and the AUCs above the MIC were 199 and 52 mg h/liter for strain 309 and 191 and 58 mg. h/ liter for strain 1209. Serum bactericidal activities and rates of killing. Following the intravenous injection of 7 mg of teicoplanin per kg or 15 mg of vancomycin per kg, no bactericidal activity could be detected at any time in the serum of rats. Figure 2 shows the rates of in vitro killing of the two E. faecalis strains tested by 32 mg of teicoplanin per liter, 36 mg of vancomycin per liter, 8 mg of gentamicin per liter, and the combination of teicoplanin and gentamicin. Neither of the E. faecalis strains showed a reduction of the bacterial counts within 48 h of exposure to peak teicoplanin or vancomycin concentrations. When exposed to gentamicin, both strains showed a 2 log1o decrease of the colony counts over 24 h. However, regrowth of the bacteria occurred at 48 h and was more pronounced for strain 309 than for strain 1209. In contrast, when exposed to a combination of teicoplanin and gentamicin, both E. faecalis strains were killed within 24 h of incubation. Prophylaxis with teicoplanin, vancomycin, and teicoplanin and gentamicin. The results of prophylaxis against the two E. faecalis strains tested are shown in Fig. 3. The ID90 was 104 CFU/ml for each E. faecalis strain. In rats challenged with this inoculum teicoplanin completely prevented endocarditis (strain 309, no rats infected; P < 10-5 versus controls). Vancomycin significantly reduced the incidence of infection
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ANTIMICROB. AGENTS CHEMOTHER.
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7mg/kg
E 4)
c)
.51 2
4
24 Hours
12
FIG. 1. Concentrations of teicoplanin, vancomycin, and gentamicin in serum in rats after a single intravenous bolus injection.
(P < 0.05 compared with controls), but prophylactic failures occurred in 8 of 20 rats (40%) challenged with E. faecalis 309 (P = 0.05 versus teicoplanin). With inocula 10 times higher than the ID90 (i.e., 105 CFU/ml), prophylaxis with teicoplanin was successful against both strains of E. faecalis (strain 309, 2 rats (13%) infected; P < 10-5; strain 1209, no rats infected; P < 10-4). Prophylaxis with vancomycin failed for both strains; 64% of the rats were infected with strain 309 (P = 0.02 versus teicoplanin), and 80% were infected with strain 1209 (P = 0.0005 versus teicoplanin). With inocula 100 times higher than the ID90 (i.e., 106 CFU/ml) vancomycin completely failed to prevent endocarditis. At this inoculum, teicoplanin almost completely protected the animals challenged with E. faecalis 1209 (1 rat of 18 was infected; P < 10-4 compared with controls; P = 0.0003 versus vancomycin). Teicoplanin also significantly prevented endocarditis in rats challenged with E. faecalis 309, but the failure rate was 37% (6 rats of 16 were infected; P = 0.001 versus controls; P = 0.008 versus vancomycin). With greater inocula (1,000 times the IDO, i.e., 107 CFU/ ml) the prophylactic effect of teicoplanin was overcome. The combination of teicoplanin plus gentamicin extended the protection conferred by teicoplanin alone to inocula 100 times the ID90 (i.e., 106 CFU/ml) in rats challenged with E. faecalis 309 (infected rats: 4% for teicoplanin plus gentamicin versus 37% for teicoplanin; P = 0.08) and to inocula 1,000 times the ID90 (i.e., 107 CFU/ml) in rats challenged with E. faecalis 1209 (infected rats: 0% for teicoplanin plus gentamicin versus 86% for teicoplanin alone; P < 10-5). Of note, for all prophylactic groups, the mean bacterial densities of the vegetations of the rats in which teicoplanin prophylaxis failed were significantly inferior to those of controls (P < 0.005). In contrast, the mean bacterial densities of the vegetations of the rats in which vancomycin prophylaxis failed were similar to those of controls.
DISCUSSION This study compared the efficacy of a single dose of teicoplanin with that of vancomycin for the prophylaxis of experimental enterococcal endocarditis in rats. For the two E. faecalis strains tested, vancomycin was only partially successful against an inoculum equal to the ID90, while teicoplanin prevented endocarditis against inocula 10 to 100 times higher than the ID90. This protection was conferred in the absence of bacterial killing. Indeed, no bactericidal activity was detected at any time in the serum of rats, and the time-kill curves did not show a significant reduction in bacterial numbers at peak concentrations of teicoplanin and vancomycin in serum. Previous studies in rats have shown that when viridans group streptococci or enterococci either were not rapidly killed or were tolerant to amoxicillin, single-dose prophylaxis of endocarditis was efficacious only at the ID90 (5). Against inocula higher than the ID90, only multiple doses of amoxicillin (viridans group streptococci) or amoxicillin plus gentamicin (enterococci) were necessary for successful prophylaxis (14). In the absence of bacterial killing, early studies (2, 7) have suggested that the inhibition of bacterial adherence to vegetations was the mechanism by which prophylaxis prevented endocarditis. However, recent observations (13, 17) have shown that prolonged inhibition of bacterial growth after the intravenous challenge, rather than inhibition of bacterial adherence, was likely to be the mechanism of protection. The longer the duration of growth inhibition is, the greater the likelihood of successful prophylaxis after challenge with inocula exceeding the ID90 is. These results suggested that prophylactic antibiotics allowed the adherent bacteria to be cleared from the valves by an as yet undetermined mechanism. Single doses of antibiotics, even when used in combination (5), have failed to protect against inocula higher the IDg, probably because blood antibiotic levels were not sustained for long enough to
TEICOPLANIN PROPHYLAXIS FOR ENTEROCOCCAL ENDOCARDITIS
VOL. 36, 1992
1259
E. faecalis 309
E-I L
0
0 2 4 6
12
24
48
Hours of incubation
E. faecalis 1209
UL
0) 0
0 2 4 6
12
24
48
Hours of incubation FIG. 2. Killing of the two E. faecalis strains tested by 32 mg of teicoplanin per liter, 36 mg of vancomycin per liter, 8 mg of gentamicin per liter, and the combination of teicoplanin and gentamicin. The antibiotic concentrations were similar to peak levels in serum obtained in rats 30 min after the intravenous injection of 7 mg of teicoplanin per kg, 15 mg of vancomycin per kg, and 4 mg of gentamicin per kg.
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ANTIMICROB. AGENTS CHEMOTHER.
ENTENZA ET AL.
E. faecalis 309 *
Log 10 MBD
7;9
:t SD
3
8.38 8.37
i0.52
-
0.69
-
*
&64 8.19 5.04
10.66
0.91
0.541
*
s85 8.96 6.25 4.24 0.40 0.60
1.07
-
9.00
6.25 4.72
103
090
0651
11 U) 0
0 -W
0 0
0 :m 0
*_
0 oc
1-
Inoculum: 103
104
1o-05
167
106
E. faecalis 1209 *
Log 10 MBD & SD
U) c
i [.
7 1.2
8
8.82 7.11
8.4
-
9
OA
L.9
8u5.56 -
5.26
1-4
3
113
1.7
.
1
0
4-.
4-0
0)
10 0-
0
I.-
*
0
22
12
Inoculum
106 105 1003
167
I
TEICOPLANIN PROPHYLAXIS FOR ENTEROCOCCAL ENDOCARDITIS
VOL. 36, 1992
1261
FIG. 3. Incidence of endocarditis in control rats (C) and in rats given single prophylactic dose of teicoplanin (T), vancomycin (V), or teicoplanin plus gentamicin (T+G), 30 min before challenge with various inocula of the two E. faecalis strains. The total number of rats is indicated at the base of each column. The mean bacterial densities (MBD) cultured from vegetations (expressed as log,( CFU per gram of vegetation) is indicated for each group. Statistical comparisons of prophylactic groups with controls: *, P < 0.05; t, P < 0.00001.
completely eliminate the bacteria attached to the vegetations. In the present study, a single intravenous dose of teicoplanin produced levels in serum which were above the MICs for the two E. faecalis strains tested for more than 12 h after the intravenous injection. In contrast, levels of vancomycin in serum were above the MIC for no longer than 4 h. Thus, it is likely that the better prophylactic effect of teicoplanin was due to a longer inhibition of bacterial growth, as shown by a greater AUC above the MIC compared with that of vancomycin (-190 versus -50 mg. h/liter). The finding that bacterial densities in vegetations of rats in which prophylaxis failed were lower in the teicoplanin groups than in either the vancomycin or the control groups supports this hypothesis. The fact that teicoplanin is retained at the periphery of the vegetations and fails to diffuse into their core (3) may contribute to its failure in the treatment of endocarditis, but as suggested by our results, this phenomenon does not impair its prophylactic efficacy, probably because the bacteria are at the surface of the vegetations not yet protected by fibrin. In conclusion, teicoplanin was superior to vancomycin for the prevention of E. faecalis endocarditis in rats. ACKNOWLEDGMENTS We thank M. Giddey and M. Knaup for technical work, A. Munafo for assistance with AUC analysis, and M. M. Wu for statistical consultation.
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