Teicoplanin and Vancomycin for Treatment of Experimental

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1991, p /91/ $02.00/0 Copyright X 1991, American Society for Microbiology Vol. 35, No. 8 Influence of Low-Level Resistance to Vancomycin on Efficacy of Teicoplanin and Vancomycin for Treatment of Experimental Endocarditis Due to Enterococcus faecium BRUNO FANTIN,1 ROLAND LECLERCQ,2 MICHEL ARTHUR,3 JEAN DUVAL,2 AND CLAUDE CARBON'* Service de Medecine Interne, Hopital Bichat, Universite Paris VII, and Institut National pour la Recherche Medicale, Unite 13, H6pital Claude Bernard, Paris,' Service de Bacteriologie-Virologie-Hygiene, H6pital Henri Mondor, Universite Paris XII, Creteil,2 and Unite des Agents Antibacteriens, Institut Pasteur, Paris Cedex,3 France Received 18 March 1991/Accepted 4 June 1991 Emergence of vancomycin-resistant strains among enterococci raises a new clinical challenge. Rabbits with aortic endocarditis were infected with Enterococcusfaecium BM4172, a clinical strain resistant to low levels of vancomycin (MIC, 16,ug/ml) and susceptible to teicoplanin (MIC, 1,Ig/ml), and against its susceptible variant E. faecium BM4172S obtained in vitro by insertional mutagenesis (MICs, 2 and 0.5,ug/ml, respectively). Control animals retained 8 to 10.5 loglo CFU/g of vegetation. We evaluated in this model the efficacy of vancomycin (30 mg/kg of body weight; mean peak and trough serum levels, 27 and 5,ug/ml, respectively), teicoplanin (standard dose, 10 mg/kg; mean peak and trough levels, 23 and 9,Ig/ml, respectively; and high dose, 20 mg/kg; mean peak and trough levels, 63 and 25,ug/ml, respectively), gentamicin (6 mg/kg; mean peak and trough levels, 8.6 and <0.1,ug/ml, respectively), alone or in combination, given every 12 h intramuscularly for 5 days. Teicoplanin standard dose was as active as vancomycin against both strains. Vancomycin was not effective against E. faecium BM4172 but was highly effective against E. faecium BM4172S (7.5 ± 1.1 log1o CFU/g of vegetation versus 4.9 ± 1.0 log1o CFU/g of vegetation for vancomycin against E. faecium BM4172 and E. faecium BM4172S, respectively; P = ). A high dose of teicoplanin was more effective than vancomycin against E. faecium BM4172 (4.4 ± 1.8 loglo CFU/g of vegetation versus 7.5 ± 1.1 loglo CFU/g of vegetation for teicoplanin high dose and vancomycin, respectively; P < 0.05). Against E. faecium BM4172 glycopeptidegentamicin combinations were the most effective regimens in vitro and in vivo (2.8 ± 0.7 and 3.5 ± 1.3 loglo CFU/g of vegetation for vancomycin plus gentamicin and teicoplanin standard dose plus gentamicin, respectively; P < 0.05 versus single-drug regimens). We concluded that high-dose teicoplanin or the combination of a glycopeptide antibiotic plus gentamicin was effective against experimental infection due to E. faecium with low-level resistance to vancomycin. Serious enterococcal infections, including bacteremia and endocarditis, are usually treated with a combination of penicillin plus an aminoglycoside (19). The glycopeptide antibiotics, vancomycin and teicoplanin, constitute the alternatives in case of bacterial resistance to beta-lactams or aminoglycosides or in patients with allergy to penicillins. Vancomycin appeared warranted by the virtual lack of enterococcal resistance to this antibiotic despite 30 years of clinical use (33). However, glycopeptide-resistant strains of enterococci responsible for colonization (21, 22, 34) or infection (31, 32) have been isolated with an increasing frequency in Europe and in the United States from patients in the presence or absence of glycopeptide therapy. Most glycopeptide-resistant strains belong to the species Enterococcus faecium and can be assigned to two phenotypes (6): high-level resistance to both vancomycin (MIC, 64 to >2,000 jig/ml) and teicoplanin (MIC,.8,ug/ml), and low-level resistance to vancomycin (MIC, 16 to 32,ug/ml) and susceptibility to teicoplanin (<1 jxg/ml). High-level resistance is often transferable and plasmid mediated (21, 22), whereas the determinant for low-level resistance to glycopeptides is assigned to a chromosomal location on the basis of lack of transfer of resistance and absence of plasmid DNA in the cell (34). The emergence of glycopeptide resistance in E. faecium, which is frequently resistant to beta-lactams (24), is a * Corresponding author cause of clinical concern since it leaves few therapeutic alternatives. A triple combination including a beta-lactam antibiotic, gentamicin, and a glycopeptide was bactericidal in vitro (20) and effective for the treatment of experimental endocarditis against strains of E. faecium with high-level resistance to vancomycin (4). However, the best therapy for the treatment of infections due to enterococci with low-level resistance to vancomycin is unknown. Teicoplanin or glycopeptide-aminoglycoside combinations could be considered as an alternative to vancomycin. The aim of this study was to evaluate the in vivo relevance of vancomycin low-level resistance and to search for an effective antimicrobial therapy of a severe experimental infection due to E. faecium with and without low-level resistance to vancomycin. For this purpose, we studied two strains of E. faecium: a clinical strain with low-level resistance to vancomycin, and a vancomycin-susceptible derivate obtained by insertional mutagenesis. The use of these isogenic strains allowed us to evaluate the in vivo relevance of low-level resistance to vancomycin and its implications on glycopeptide efficacy. MATERIALS AND METHODS Organisms. E. faecium BM4172 was isolated in 1990 from the stool of a patient who previously received oral decontamination including vancomycin. This strain was identified as an enterococcus by absence of catalase, inability to

2 VOL. 35, 1991 TREATMENT FOR ENDOCARDITIS DUE TO E. FAECIUM 1571 produce gas, presence of Lancefield antigen group D, and growth on 40% bile in 6.5% sodium chloride-0.1% methylene blue and at ph 9.6. Species identification (8, 28) was based on the absence of reduction of potassium tellurite and tests for acid production from 50 carbohydrates in API 50 CH galleries (API, la Balme-les-Grottes, France). The vancomycin-susceptible strain E. faecium BM4172S was constructed by insertional mutagenesis of E. faecium BM4172 with a suicide vector that exploited the transposition properties of TnJ545 (30). Detailed description of the construction of E. faecium BM4172S and characterization of the gene inactivated by insertion of the suicide vector will be described elsewhere (9). E. faecium BM4172 and E. faecium BM4172S were indistinguishable in API 50 CH galleries. No mutant with increased MICs of glycopeptides could be selected after plating 109 CFU of E. faecium BM4172 and E. faecium BM4172S on agar plates containing serial dilutions of teicoplanin or vancomycin. The stability of vancomycin resistance inactivation in E. faecium BM4172S was checked by determination of glycopeptide MICs after seven consecutive overnight subcultures in antibiotic-free Mueller-Hinton broth (MHB). Media and antibiotics. MHB and Mueller-Hinton agar (Diagnostics Pasteur, Marnes-la-Coquette, France) were used. All incubations were at 37 C. Drugs were supplied by their manufacturers; gentamicin, Unilabo, Levallois-Perret, France; teicoplanin, Merrell Dow, Levallois-Perret, France; vancomycin, Eli Lilly & Co., Saint-Cloud, France. In vitro susceptibility to antibiotics. MICs and MBCs of gentamicin, teicoplanin, and vancomycin were determined by the macrodilution method, with inocula of 5 x 105 and 5 x 107 CFU/ml, to test the susceptibilities under standard conditions and to reproduce the high bacterial concentration present in vegetations (12). The MIC was defined as the lowest concentration of antibiotic that prevented turbidity after 24 h of incubation. The MBC was defined as the lowest concentration of antimicrobial agent that killed at least 99.9% of the original inoculum (26). The influence of serum on teicoplanin susceptibility was determined by MIC performed in 50% MHB and 50% normal serum rabbit. Study of combined antimicrobial activity. Time-kill curves were used to test the bactericidal activities of gentamicin, teicoplanin, and vancomycin, alone or in combinations (20). Overnight cultures were diluted in glass tubes containing 10 ml of fresh MHB to yield an inoculum of approximately 5 x 106 CFU/ml. The following concentrations were used: 4,ug/ml of gentamicin, alone or in combinations with various glycopeptide concentrations; 1, 8, and 30,ug/ml of teicoplanin; and 8 and 30 jig/ml of vancomycin. For every drug, the concentrations used were within clinically achievable ranges. After 0, 3, 6, 24, and 48 h of incubation, aliquots were plated on Mueller-Hinton agar. The agar plates were incubated for 24 h before CFUs were counted. In preliminary experiments, antibiotic carryover was ruled out by plating samples of bacterial suspensions containing 101 and 103 CFU/ml in the presence or absence of glycopeptide antibiotics alone or in combination with gentamicin (26). Bactericidal activity was defined as an at least 3 log1o decrease in the original inoculum. Experimental endocarditis. Investigations were performed in female New Zealand White rabbits (weight range, 1.8 to 2.5 kg). Aortic endocarditis was induced by insertion of a polyethylene catheter through the right carotid artery into the left ventricle to induce the formation of vegetations, as previously described (11). Twenty-four hours after catheter insertion, each rabbit was inoculated by ear vein with 108 CFU of E. faecium (mean, 2 x 108 CFU; range, 1 x 108 to 5 x 108 CFU) in 1 ml of 0.9% NaCl. This inoculum produced endocarditis in all rabbits with proper placement of the catheter. The catheter was left in place throughout the experiment. Forty-eight hours after inoculation, animals received intramuscularly every 12 h one of the following regimens for 5 days: vancomycin, 30 mg/kg of body weight; teicoplanin, 10 mg/kg after a loading dose of 20 mg/kg (standard dose); teicoplanin, 20 mg/kg after a loading dose of 40 mg/kg (high dose); gentamicin, 6 mg/kg; vancomycin plus gentamicin; or teicoplanin standard dose plus gentamicin. Control animals were left untreated. Those regimens were chosen according to the literature because they reproduced levels in serum comparable to those obtained in humans. Blood was sampled 1 h after antibiotic injection on day 5 of antimicrobial therapy and at the time of sacrifice (i.e., 12 h after the last antibiotic dose) for determination of peak and trough antibiotic levels in animals treated with a single antimicrobial agent. 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. Seventy-three of 100 animals had evidence of macroscopic vegetations at the time of sacrifice and generated in vivo data. All vegetations from each rabbit were excised, pooled, and weighted. The vegetations from each rabbit weighed a total of 10.2 to 69.4 mg. They were homogenized in 0.5 ml of sterile saline, and 0.1-ml portions were quantitatively subcultured onto agar plates for 24 h. Colony count results were expressed as log1o CFU per gram of vegetation. Vegetations were considered sterile when no growth occurred from a 0.1-ml subculture of the undiluted homogenate. For the calculation of the mean number of CFU per gram of vegetation, such vegetations were considered to contain a maximum of 1 CFU, which corresponded to approximately 2.0 to 2.5 log1o CFU/g, according to the weight of the vegetations. After a 1:10 suspension of each vegetation in saline, 0.1-ml portions were plated onto agar plates containing vancomycin or teicoplanin in a final concentration of 1 and 2 times the MIC of each glycopeptide for the corresponding strain to detect selection of mutant resistant strain. Antibiotic carryover. Antibiotic carryover was tested when antibiotic concentrations in vegetation homogenates were above the MIC of the antibiotic for the study strain at the dilution used to accurately count the CFU per plate (>20 CFU per plate). This was done by plating samples of bacterial suspension containing 101 to 103 CFU/ml of MHB in the absence or presence of antibiotic at a concentration equal to that observed in the vegetation homogenate. Antibiotic assays. Antibiotic concentrations were measured in serum and in vegetations of animals by the agar diffusion method (10). Indicator organism was Bacillus subtilis ATCC 6633 (Difco Laboratories, Detroit, Mich.), and media were antibiotic medium no. 1 (Difco) for teicoplanin and vancomycin and antibiotic medium no. 5 for gentamicin. The sensitivity of the assay was 0.1, 1, and 0.5,ug/ml for gentamicin, teicoplanin, and vancomycin, respectively. Some serum specimens containing teicoplanin were also assayed by high-performance liquid chromatography, as described by Jehl et al. (17). Statistics. The bacterial concentrations in vegetations from the various groups of animals infected with the same strain were compared by an analysis of variance followed by the Scheffe test for multiple comparisons. Comparisons of bacterial concentrations in vegetations from two groups of

3 1572 FANTIN ET AL. TABLE 1. Antibiotic MICs and MBCs of antibiotics against E. faecium BM4172 and BM41725 MIC/MBC (ug/ml) against E. faecium BM4172 E. faecium BM x x x x 107 CFU/ml CFU/ml CFU/ml CFU/ml Vancomycina 16/32 16/32 2/32 4/32 Teicoplanina 1/16 4/32 0.5/16 2/16 Gentamicin 16/16 16/16 16/16 16/16 a MBCs after 48 h of incubation (>128,g/ml after 24 h of incubation). animals infected with different strains and treated with the same regimen were done by an unpaired t test. A P value of <0.05 was considered significant. All the results are expressed as means + standard deviations. RESULTS In vitro susceptibility to antibiotics. The MICs and MBCs of antimicrobial agents for the two strains are shown in Table 1. Insertional inactivation resulted in a eightfold and a twofold decrease in MICs of vancomycin and teicoplanin, respectively. Although not very impressive, the one-dilution decrease in MIC of teicoplanin was consistently observed on three determinations. A 2 log1o CFU/ml increase in the size of the inoculum produced a fourfold increase in MIC for teicoplanin, whereas the activity of gentamicin and vancomycin were not significantly affected by the size of the inoculum. Thus, at the highest inoculum tested, vancomycin MICs were only fourfold and twofold greater than teicoplanin MICs, for E. faecium BM4172 and E. faecium BM4172S, respectively. Addition of rabbit serum resulted in a twofold increase in MIC for teicoplanin (MIC = 2,ug/ml for E. faecium BM4172 with a standard inoculum in MHB plus serum versus 1,ug/ml in MHB alone). The two strains had a low-level resistance to gentamicin (MIC, 16,ug/ml). Glycopeptide MBCs, as shown in Table 1, were all determined after 48 h of incubation, since no significant bactericidal effect was observed after 24 h of incubation (MBC, >128,ug/ml). The MIC of penicillin for BM4172 was 1,ug/ml. Study of combined antimicrobial activity. The lack of significant bactericidal activity for glycopeptides during the first 24 h of incubation was confirmed by the killing curves (Fig. 1). For E. faecium BM4172 (Fig. 1A), vancomycin or teicoplanin alone were bactericidal only after 48 h of incubation and with the highest concentration tested (30,ug/ml), which corresponded to approximately 2 times the MIC of vancomycin and 30 times the MIC of teicoplanin. There was a 2.0 log1o CFU/ml increase in killing with teicoplanin at 30,ug/ml versus 8,ug/ml, concentrations which corresponded to the mean levels achieved at trough in serum of rabbits treated with the high or the standard dose of teicoplanin, respectively. The combination of vancomycin used in subinhibitory concentrations and gentamicin was ANTIMICROB. AGENTS CHEMOTHER. not synergistic. In contrast, an enhanced bactericidal effect with the aminoglycoside was observed when the glycopeptide antibiotics were tested at inhibitory concentrations, after 24 h of incubation for vancomycin at 30 p.g/ml and teicoplanin at 30,ug/ml, and after 48 h of incubation for only teicoplanin at 1 or 8 jxg/ml. At inhibitory concentrations, glycopeptide behavior on E. faecium BM4172S was very similar to that against E. faecium BM4172 (Fig. 1B). Again, only the highest concentration tested (30,ug/ml) achieved bactericidal activity after 48 h of incubation. Combination with gentamicin resulted in significant bactericidal activity after 48 h of incubation when inhibitory concentrations of teicoplanin (1 and 8 mg/liter) and vancomycin (8,ug/ml) were used (data not shown). As with E. faecium BM4172, the only bactericidal regimens after 24 h of incubation were teicoplanin or vancomycin at 30,ug/ml in combination with gentamicin. Antibiotic levels in serum and vegetations. Antibiotic serum levels are shown in Table 2. All the values were in the range of achievable concentrations in humans. Peak levels of teicoplanin standard dose in serum measured by high-performance liquid chromatography (HPLC) were comparable to those obtained by the microbiologic method (21 ± 6 versus 23 ± 5 jig/ml [n = 4], for HPLC and microbiologic method, respectively). Gentamicin and vancomycin were not detectable in the undiluted vegetation homogenate; in contrast, teicoplanin concentrations in vegetations (n = 8) were 24.2 ± 6.3 and 68.4 ± 17.9 j,g/g for the standard and the high-dose regimen, respectively. Experimental endocarditis. In vivo results are shown in Table 3. Gentamicin, vancomycin, and teicoplanin standard dose were not significantly more effective than controls against the vancomycin-resistant strain. Teicoplanin high dose was the only single-drug therapy to exhibit significant efficacy versus controls (P < 0.01) and vancomycin (P < 0.05). Combination therapies were the most effective treatments. Vancomycin plus gentamicin produced a 4.7 log1o CFU/g of vegetation-greater reduction than vancomycin alone (P < 0.01) and teicoplanin plus gentamicin induced a 3.5 log1o CFU/g of vegetation-greater reduction than teicoplanin alone (P < 0.01). Only animals treated with combination therapies retained sterile vegetations. Vancomycin was highly effective against E. faecium BM41725 (P < 0.01)-more effective than against E. faecium BM4172 (P = ). Teicoplanin standard dose was as effective as vancomycin against E. faecium BM4172S but more effective than vancomycin against E. faecium BM4172 (P = 0.034). When the data were expressed in terms of mean bacterial reduction in comparison with controls, the differences between the two strains were even more pronounced, since bacterial titers in vegetations of the control groups tended to be higher, although nonsignificantly (P = ), for E. faecium BM4172S than for E. faecium BM4172 (-2.0 versus -5.2 log1o CFU/g of vegetation-greater reduction than controls for vancomycin [P = ] and -2.5 versus -4.4 log1o CFU/g of vegetation-greater reduction than controls for teicoplanin [P = ] for E. faecium BM4172 and E. faecium BM4172S, respectively). No mutant strains with increased glycopeptide MICs could be recovered from vegetations. Carryover. Since vancomycin and gentamicin residual concentrations were not detectable in vegetations, an antibiotic carryover effect could be ruled out for the regimens including those antibiotics. For teicoplanin standard dose, concentrations in the undiluted vegetation homogenates were 2.6 ± 0.5,ug/ml; since a 10-1 (or even greater) dilution of the original homogenates was always necessary to accurately determine the bacterial counts in vegetations, teicoplanin concentrations were always below the MIC at this dilution (0.26,ug/ml for an MIC of 0.5,ug/ml). The only regimen for which carryover might be relevant was teicoplanin high dose. In this experiment, teicoplanin concentrations

4 VOL. 35, 1991 A cfu/ml TREATMENT FOR ENDOCARDITIS DUE TO E. FAECIUM 1573 Control G V8 io6 101 B cfu/ml Hours V8G TI T8 TI G T30 V30 T30G V30 G Control =:Q G V8 Ti T8 T30 V Hours FIG. 1. In vitro killing rates in MHB of E. faecium BM4172 (A) and E. faecium BM4172S (B), incubated with gentamicin (G; 4,g/ml), teicoplanin (Ti, T8, T30 [1, 8 and 30,ug/ml, respectively]), and vancomycin (V8 and V30 [8 and 30,ug/ml, respectively]) alone or in combinations. The control was antibiotic-free medium. in the undiluted vegetation homogenate were ,ug/ml for an MIC of 1,ug/ml. In three of eight vegetations, the CFU could only be counted for the undiluted portion of the homogenate. However, no carryover effect was observed with the technique described in Materials and Methods with 5 and even 10,ug of teicoplanin per ml; a significant carryover effect was only noted for concentrations of teicoplanin equal to 20 jxg/ml.

5 1574 FANTIN ET AL. TABLE 2. Antibiotic concentrations in the sera of rabbits infected with E. faecium after 5 days of treatment Antibiotic (n) Regimen (mg/kg, Mean + SD concn (,ug/ml) twice per day) Peak (1 h) Trough (12 h) Vancomycin (5) ± 12 5 ± 3 Teicoplanin (4) loa 23 ± 5 9 ± 1 Teicoplanin (4) 20b 63 ± ± 10 Gentamicin (4) ± 2.2 <0.1 a After a loading dose of 20 mg/kg. "After a loading dose of 40 mg/kg. DISCUSSION The purpose of this study was to evaluate the in vivo relevance of vancomycin low-level resistance and to search for an effective antimicrobial therapy of a severe experimental infection due to E. faecium with and without low-level resistance to vancomycin. We were able to demonstrate, using the aortic valve endocarditis model in rabbits, that a low-level resistance to vancomycin was responsible for therapeutic failure of vancomycin alone. This fact is of major clinical importance, since low-level resistance to vancomycin is difficult to detect (27) and thus might be underestimated (20). Vancomycin failure could not be attributed to an inappropriate dosing regimen for two reasons: (i) vancomycin levels in serum were comparable to those achieved in humans, with a peak of around 30 Fxg/ml and a trough of around 5 p,g/ml (Table 2); and (ii) the same vancomycin regimen was very effective against a susceptible variant, obtained after insertional mutagenesis, which was as virulent as the resistant parent (Table 3). The use of these isogenic strains led us to conclude that in vivo failure of vancomycin was related to expression of low-level vancomycin resistance. Since the currently recommended MIC breakpoints for vancomycin are 32 and 4,ug/ml (1), E. faecium BM4172 was considered as intermediate to vancomycin. Our results indicated that vancomycin therapy was not effective against the intermediate enterococcal strains. The report of a clinical failure of vancomycin in the treatment of peritoneal infection due to a strain of Staphylococcus haemolyticus with an MIC of 8 plg/ml is consistent with our observation (29). Low-level resistance to vancomycin among enterococi could theoretically be an indication for the use of teicoplanin, since the strains remain susceptible to the latter drug, with MICs of <1,ug/ml (18). Teicoplanin is a glycopeptide antibiotic available for several years in Europe, with pro- TABLE 3. Results of different 5-day treatment regimens in rabbits infected with E. faecium strains Treatment Log1o CFU/g of vegetation (mean e SD) (no. sterile/total vegetations)a E. E. faecium BM4172 E. ~~~~BM41725 faecium Control 9.5 ± 0.8 (0/7) 10.1 ± 0.2 (0/8) Gentamicin 7.9 ± 2.4 (0/8) ND Vancomycin 7.5 ± 1.1 (0/6) 4.9 ± 1.0 (0/7)* Teicoplanin 7.0 ± 1.1 (0/8) 5.7 ± 1.1 (0/8)* Teicoplanin high dose 4.4 ± 1.8 (0/8)*t ND Vancomycin + gentamicin 2.8 ± 0.7 (2/5)*tt ND Teicoplanin + gentamicin 3.5 ± 1.3 (3/8)*tt ND I ND, not done; *, more effective than control (P < 0.001); t, more effective than vancomycin (P < 0.05); t, more effective than teicoplanin (P < 0.01). ANTIMICROB. AGENTS CHEMOTHER. longed serum half-life allowing once-daily dosing (2). With the standard dose of teicoplanin used in our experiments, we reproduced in rabbits levels in serum comparable to those achieved in humans with the currently recommended regimen (6 mg/kg/day after a 24- to 48-h loading dose period [13]). However, this regimen was not significantly more effective than vancomycin. Several pharmacodynamic and pharmacokinetic parameters might account for the discrepancy between favorable MICs and in vivo results with teicoplanin: (i) high MBC-MIC ratio, as observed in our study and confirmed by a low rate of killing in vitro with concentrations equal to or below 8,ug/ml; (ii) influence of the size of the inoculum (so-called inoculum effect [16], a phenomenon which was observed in our in vitro study and is particularly important to consider when one deals with left-sided endocarditis, an infection with a high bacterial concentration in vegetation); (iii) high protein binding (:90% protein binding to rabbit serum for teicoplanin [5] versus :65% for vancomycin [25]), a parameter which clearly affects the in vivo MIC, as shown in our study; and (iv) low penetration into the core of the infected vegetation, as recently shown by quantitative autoradiography (7), despite high concentrations in the vegetation homogenate. Against the vancomycin-susceptible strain, in contrast, teicoplanin exhibited good in vivo efficacy, comparable to that of vancomycin, and better than the same regimen against the vancomycin-resistant parent. This result is of great interest, because it was obtained with an isogenic couple of strains, the only parameter differing from one experiment to another being the MIC of teicoplanin (1 versus 0.5 pg/ml for the vancomycin-resistant and susceptible strain, respectively). Although this difference was not very impressive, it was consistently observed in vitro. When one considers the combined influence of a high inoculum and protein binding, two factors which are important to take into account in vivo and clearly affect teicoplanin activity as shown by our in vitro data (fourfold increase in MIC with the higher inoculum, and twofold increase in MIC when tested in the presence of rabbit serum), then the inhibitory concentrations in vivo for teicoplanin against our two study strains might be.8 and 4 pug/ml, for the vancomycin-resistant and susceptible strains, respectively. In this range of concentrations, this difference may be relevant in vivo when teicoplanin trough serum levels are just above 8 alg/ml (Table 2). There is a growing evidence in the clinical and experimental literature that high concentrations of teicoplanin are required to achieve efficacy in vivo (3, 13-15, 23, 35). Indeed, in our experimental study, the high teicoplanin dosage was the only single-drug regimen to significantly achieve bactericidal activity against E. faecium BM4172. This good efficacy confirmed our in vitro findings, since the highest concentration of teicoplanin tested (30,ug/ml) was the only one to achieve in vitro bactericidal activity after 48 h of incubation; this concentration corresponded approximately to the trough level in serum achieved in rabbits treated with teicoplanin high dose regimen (Table 2). An important result in our study is the observation of an enhanced bactericidal activity in vivo between a glycopeptide antibiotic and gentamicin, even on a strain with lowlevel resistance to vancomycin. This result was in accordance with our killing curves performed with inhibitory but not bactericidal concentrations of glycopeptides. In conclusion, we demonstrated the in vivo relevance of vancomycin low-level resistance for an E. faecium strain, responsible for therapeutic failure in a severe experimental infection. A standard dose of teicoplanin was as active as

6 VOL. 35, 1991 TREATMENT FOR ENDOCARDITIS DUE TO E. FAECIUM 1575 vancomycin against a relatively vancomycin-resistant strain and the vancomycin-susceptible derivate obtained after insertional mutagenesis. Teicoplanin had to be used in a high-dosing regimen to achieve in vivo benefit in comparison with vancomycin, despite a low MIC for the latter. Vancomycin and teicoplanin exhibited an enhanced bactericidal effect in vitro and in vivo in combination with aminoglycoside despite low-level resistance to vancomycin. ACKNOWLEDGMENTS This work was supported in part by Institut National de la Santd et de la Recherche Medicale, Unitd 13, by Universitd Paris VII, and by a grant from Merrell Dow France. We thank J. P. Di Piazza for technical assistance and N. Muller for secretarial assistance. REFERENCES 1. Amsterdam, D The new National Committee for Clinical Laboratory Standards susceptibility documents. Antimicrob. Newsl. 6: Buniva, G., A. Del Favero, A. Bernareggi, L. Patoia, and R. 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