ANTIMICROBIAL AGENTS AN CHEMOTHERAPY, Sept. 12, p. 1-1 -/2/1-$2./ Copyright C 12, American Society for Microbiology Vol., No. Comparison of aptomycin, Vancomycin, and Ampicillin- Gentamicin for Treatment of Experimental Endocarditis Caused by Penicillin-Resistant Enterococci MARCELO C. RAMOS,lt M. LINSAY GRAYSON,2 t GEORGE M. ELIOPOULOS,2 AN ARNOL S. BAYER' * ivision of Infectious iseases, Harbor- University of California, Los Angeles, Medical Center, Torrance, California 1; ivision of Infectious iseases, New England eaconess Hospital2 and Harvard Medical School, Boston, Massachusetts 221; and School of Medicine, University of California, Los Angeles, Los Angeles, California 2 Received 2 April 12/Accepted 2 June 12 Infections with enterococci that are resistant to multiple antibiotics are an emerging clinical problem. We evaluated the antibiotic treatment of experimental enterococcal endocarditis caused by two strains with different mechanisms of penicillin resistance. Enterococcusfaecalis HH-22 is resistant to aminoglycosides and penicillin on the basis of plasmid-mediated modifying enzymes; Enterococcus raffinosus SF-1 is susceptible to aminoglycosides but is resistant to penicillin on the basis of low-affinity penicillin-binding proteins. Animals infected with strain HH-22 received days of treatment with the following: no treatment; daptomycin (2 mg/kg of body weight twice daily [b.i.d.], intramuscularly [i.m.]), vancomycin (2 mg/kg b.i.d., intravenously), or ampicillin (1 mg/kg three times daily, i.m.) plus gentamicin (2. mg/kg b.i.d. i.m.). Although vancomycin was superior to ampicillin-gentamicin (P <.1), daptomycin was significantly better than all other treatment regimens (P <.1) in reducing intravegetation enterococcal densities, although no vegetations were rendered culture negative by this agent. Animals infected with strain SF-1 received days of no therapy, ampicillin, ampicillin-gentamicin, vancomycin, or daptomycin (all at the dosage regimens described above). aptomycin, vancomycin, and ampicillin-gentamicin each lowered intravegetation enterococcal densities significantly better than did ampicillin monotherapy or no treatment (P <.1); moreover, these three treatment regimens rendered significantly more vegetations culture negative than did ampicillin monotherapy or no treatment (P <.). Serum daptomycin levels remained above the MICs and MBCs for both enterococcal strains throughout the 12-h dosing interval used in the study. aptomycin and vancomycin were both active in vivo in these models of experimental enterococcal endocarditis caused by penicillin-resistant strains, irrespective of the mechanism of resistance. This activity correlated with the unique cell wall sites of action of these agents (binding to lipoteichoic acid and pentapeptide precursor, respectively) compared with the sites of action of 1-lactams (penicillin-binding proteins). 1-Lactamase production by strain HH-22 precluded in vivo efficacy with ampicillin-gentamicin combinations. In contrast, this combination was active in vivo against strain SF-1, which exhibited intermediate-level penicillin resistance (MIC, 2,ug/ml), likely reflecting the ability of high-dose ampicillin to achieve enough binding to low-affinity penicillin-binding proteins to cause augmented aminoglycoside uptake. In the past decade there has been an emergence of enterococcal strains that have developed resistance to a variety of cell wall-active agents (including penicillins and vancomycin) as well as to most aminoglycosides (,, 1, 1-22). These include Enterococcusfaecalis strains that are resistant to aminoglycosides by virtue of plasmid-mediated production of modifying enzymes and that may concomitantly possess either plasmid-mediated (1, 1, 1, 22) or chromosomally mediated means of producing P-lactamase (2). Moreover, recent outbreaks of penicillin-resistant enterococcal infections have been identified. These outbreaks were related to I-lactamase-negative strains. -Lactamase-negative strains have most commonly been non- * Corresponding author. t Present address: Faculty of Medicine, State University of Campinas, Campinas, Brazil. t Present address: Monash Medical Centre, Melbourne, Australia. E. faecalis enterococci (e.g., E. faecium and E. raffinosus [1, 21]). For optimal efficacy, traditional treatment for deep-seated enterococcal infections, such as endocarditis, requires combination chemotherapy with a cell wall-active agent (e.g., penicillin and vancomycin) plus an aminoglycoside. The basis for this synergistic effect is augmented aminoglycoside uptake in the presence of the cell wall-active agent (1). The presence of penicillin-resistant enterococci in a deep-seated infection such as endocarditis would severely limit the therapeutic options. The study described here was designed to compare the in vivo effects of daptomycin, a cyclic lipopeptide agent with potent antienterococcal activity, versus those of standard antienterococcal regimens, including vancomycin and ampicillin (with or without aminoglycosides). The experimental model of penicillin-resistant enterococcal endocarditis was used as a rigorous test of antimicrobial efficacy, with animals infected with either a,b-lactamase-producing, aminoglycoside-resistant strain of E. faecalis or a,-lactamasenegative, aminoglycoside-susceptible strain of E. raffinosus. ownloaded from http://aac.asm.org/ on November, 21 by guest 1
VOL., 12 PENICILLIN-RESISTANT ENTEROCOCCAL ENOCARITIS 1 MATERIALS AN METHOS Bacterial strains. E. faecalis HH-22 was kindly provided by Barbara Murray (Houston, Tex.); this strain has been described in detail elsewhere (1). E. faecalis HH-22 possesses plasmid-mediated penicillin and aminoglycoside resistance. E. raffinosus SF-1, a,b-lactamase-negative, penicillin-resistant clinical isolate was kindly provided by Kathryn L. Rouff (Boston, Mass.). For all studies, enterococcal strains were grown overnight in cation-supplemented Mueller-Hinton broth (CSMHB; ifco Laboratories, etroit, Mich.) containing meq of calcium and 2 meq of magnesium; they were then isolated for purity after 1 h of growth on % sheep blood agar. Antibiotics. aptomycin was provided by Eli Lilly Research Laboratories, Indianapolis, Ind., while vancomycin, ampicillin, penicillin G, and gentamicin were purchased from commercial sources. All agents were reconstituted according to the manufacturers' package inserts or recommendations. In vitro studies. The MICs and MBCs of daptomycin, vancomycin, ampicillin, penicillin G, and gentamicin for the enterococcal isolates were determined by the broth microdilution technique by using logarithmic-phase organisms in CSMHB at a final inoculum of 1 CFU/ml. The MICs and MBCs of daptomycin were determined in parallel in the presence of 1 or % rabbit serum because of the high protein binding of this compound (). For each determination, the MIC was defined as the lowest antibiotic concentration that prevented visible turbidity after 2 h of incubation at C. The MBC was defined as the lowest antibiotic concentration that yielded >.% killing of the final inoculum, as determined by quantitatively culturing -pr samples from all clear microtiter wells onto sheep blood agar and incubating them for 2 h at C. For MBC testing at this inoculum, the rejection value involved a single surviving colony; thus, we were at the sensitivity limit of the assay. Susceptibility testing was also done in parallel with Staphylococcus aureus ATCC 22, which was used as an internal quality control. The MICs and MBCs of the study drugs for this organism are known. The enterococcal strains were also evaluated by the time-kill curve technique for susceptibility to the study antibiotics; they were tested at a final inoculum of -1 enterococcal CFU/ml, reflecting a bacterial density achieved early after the induction of experimental endocarditis (). The antibiotic concentrations used in the time-kill studies represented levels that are readily achievable in rabbit sera following treatment with the dosage regimens used in this investigation (1, 2, ). The enterococcal strains at the appropriate inoculum were added to antibiotic-containing CSMHB to achieve the following final drug concentrations: daptomycin, or jig/ml; ampicillin, 1 or 2,g/ml; vancomycin, 1 or,g/ml; gentamicin,,g/ml. Also, for daptomycin, parallel kill curves were determined in the presence of 1 or % rabbit serum, as described above. Samples of 1,ul were drawn at,, and 2 h of incubation at C and were quantitatively subcultured onto blood agar plates for an additional 2 h at C. Kill curves were then constructed by comparing bacterial survival (log1o CFU per milliliter) versus time. For the drug combination ampicillin-gentamicin, bactericidal synergy was defined as a.2 log1 CFU/ml difference between the CFU/ml of the cultures treated with drug combination compared with the CFU/ml of the cultures treated with the most active of the single drugs in the combination. Antibiotic levels in serum. The pharmacokinetics of ampicillin, gentamicin, and vancomycin in rabbits have been well characterized by us and others (1, ), and so they were not repeated. To delineate the postdose levels of daptomycin in serum over time, five healthy female New Zealand White rabbits were injected intramuscularly (i.m.) with 2 mg of daptomycin per kg of body weight; blood samples were then drawn at,, 12, 1, and 2 min and 12 h postdose (representing the 12-h dosing interval). aptomycin levels in serum were kindly determined by James Woodworth (Eli Lilly Research Laboratories) by a well-diffusion microbiological assay by using Micrococcus luteus ATCC 1 as the test organism; the sensitivity of this assay is.1 ug/nml. Animal experiments. The experimental rabbit model was used to assess the therapeutic efficacies of the study drugs against the two enterococcal strains. Anesthetized, female New Zealand White rabbits underwent transaortic catheterization as described previously (1), and then 2 h later they were infected with -1 CFU of either enterococcal strain HH-22 or enterococcal strain SF-1 via the marginal ear vein; this challenge inoculum caused endocarditis in >% of catheterized animals in pilot studies in our laboratory. Catheters remained in place for the duration of the study. Blood cultures were performed at 2 h postbacterial challenge, and bacteremic animals were randomized to receive daptomycin (2 mg/kg twice daily, [b.i.d.], i.m.), vancomycin (2 mg/kg b.i.d., intravenously), and ampicillin (1 mg/kg three times daily [t.i.d.], i.m.) with or without gentamicin (2. mg/kg b.i.d., i.m.). Animals infected with the P-lactamase-producing, aminoglycoside-resistant strain (HH-22) did not receive ampicillin or gentamicin monotherapy because of the documented ineffectiveness of these agents against this strain in vivo (). Similarly, animals infected with the aminoglycoside-susceptible strain (SF-1) did not receive gentamicin monotherapy, despite the in vitro susceptibility of this strain to gentamicin; pilot studies in our laboratory indicated that gentamicin monotherapy is ineffective in this model. After days of therapy, the surviving rabbits were euthanized; aortic valve and left ventricle vegetations from individual animals were removed, pooled, weighed, homogenized in. ml of sterile saline solution, and quantitatively cultured in CSMH agar (ifco Laboratories). Sacrifices were performed at least 2 h after the last antibiotic dose; this, in combination with serial dilution of the vegetation homogenates (21-) prior to subculture, minimizes antibiotic carryover effects. For purposes of statistical analyses, culture-negative vegetations were considered to contain c2 log1o CFU/g () because of the small average vegetation weights (-.1 g) and the initial dilution of the vegetations prior to the homogenizations. Statistical analysis. The mean + standard deviation bacterial densities per gram of vegetation in the various treatment groups were compared for the two enterococcal strains by analysis of variance. The Bonferroni factor and the Student- Newman-Kuel tests were done to correct for multiple comparison groups. Fisher's exact test was used to compare proportional data. AP value of <. was used to designate significant differences. RESULTS In vitro studies. The MICs and MBCs of the study drugs for the two enterococcal strains at an inoculum of _1 CFU/ml are listed in Table 1. Strain HH-22 was susceptible to daptomycin and vancomycin, but was resistant to gentamicin. Of note, this strain appeared to be susceptible to ownloaded from http://aac.asm.org/ on November, 21 by guest
1 RAMOS ET AL. TABLE 1. In vitro susceptibilities of enterococcal strains HH-22 and SF-1a MIC/MBC (,ug/ml) Agent E. faecalis E. raffinosus HH-22 SF-1 aptomycin 1/./ Vancomycin 2/2./1 Penicillin / 2/12 Ampicillin 2/2 1/2 Gentamicin >2/>2./1 a An inoculum of -1 CFU/ml in a microtiter system was used. ampicillin and penicillin at an inoculum of _1 CFU/ml (MICs, 2 and p,g/ml, respectively); however, the expression of,-lactamase resistance for this strain is manifest at higher bacterial inocula, as seen within cardiac vegetations (e.g., ampicillin MIC and MBC, 2 and >12,g/ml, respectively, at an inoculum of -1 CFU/ml [1]). Strain SF-1 was susceptible to daptomycin, vancomycin, and gentamicin but was resistant to penicillin G (MIC, 2,ug/ml) and ampicillin (MIC, 1,ug/ml). _ By the time-kill curve technique at an inoculum of 1 CFU/ml, daptomycin at,ug/ml (but not,g/ml) was rapidly bactericidal against strain HH-22 (Fig. 1) in the presence or absence of 1 or % rabbit serum. In contrast, vancomycin was only slowly bactericidal in vitro against this strain, with an -2 log1o decline in CFU per milliliter by 2 h of incubation. As anticipated, gentamicin was not effective against strain HH-22 in vitro, while ampicillin alone (1 or 2,ug/ml) or in combination with gentamicin was not rapidly bactericidal against this strain (Fig. 2). aptomycin was also active in vitro against strain SF-1 (Fig. ). Similar to strain HH-22, there was no substantial interference with daptomy- E C -J 1 2 / < 1 2 _ 1 ANTIMICROB. AGENTS CHEMOTHER. 2 FIG. 2. Time-kill curves showing the in vitro activities of ampicillin at 1 (V) or 2 (A),ug/ml, ampicillin at 1 ±g/ml in the presence of gentamicin at p,g/ml (M), and gentamicin at pg/ml () against E. faecalis HH-22., control. cin's activity against SF-1 by the addition of 1 or % rabbit serum (data not shown); vancomycin was less active than daptomycin against strain SF-1. While ampicillin alone at either 1 or 2 p,g/ml was not active against strain SF-1, the combination of ampicillin-gentamicin was substantially bactericidal (Fig. ); however, the excellent bac- La C -J - ownloaded from http://aac.asm.org/ on November, 21 by guest 2 FIG. 1. Time-kill curves showing the in vitro activities of daptomycin and vancomycin against E. faecalis HH-22. *, control; *, daptomycin at,ug/ml in the presence of % rabbit serum; A, daptomycin at,ug/ml;, vancomycin; *, daptomycin at,ug/ml; V, daptomycin at,g/ml in the presence of % rabbit serum. 2 FIG.. / 2 Time-kill curve showing the in vitro activities of daptomycin at (O) and (),ug/ml versus those of vancomycin at 1 (V) and (),g/ml against enterococcal strain E. raffinosus SF-1. A, control.
VOL., 12 PENICILLIN-RESISTANT ENTEROCOCCAL ENOCARITIS 1 E I 1-2 2 FIG.. Time-kill curve showing the in vitro activities of ampicillin alone at 1 (V) or 2 (El),ug/ml versus those of ampicillin (1 plg/ml)-gentamicin ( 1±g/ml) () and gentamicin alone at,ug/ml () against E. raffinosus SF-1. A, control. tericidal activity of gentamicin alone against this strain precluded demonstration of bactericidal synergy at the drug concentrations that we used. Antibiotic levels in serum. As noted in Fig., the mean levels of daptomycin in serum were >1,ug/ml for at least h postdose and were well above the MICs and MBCs for both enterococcal strains throughout the 12-h dosing interval Ȧnimal endocarditis studies. For animals infected with E. faecalis HH-22, days of daptomycin therapy was highly effective at reducing vegetation bacterial densities to signif- 2 1 E 1 L 1.' 12 E 1 1 X 2 1 2 1 11 12 Time (hours) FIG.. Serum daptomycin levels over a 12-h dosing interval following a 2-mg/kg dose (i.m.). Bars represent standard deviations. v TABLE 2. aptomycin compared with vancomycin and ampicillin-gentamicin for therapy of experimental enterococcal endocarditis caused by E. faecalis HH-22 Regimen NNo. of Postinfection Mean +F S of vegetation aptomycin (2 mg/ 1.1 ± 1.2a. kg b.i.d.) Vancomycin (2 mg/ 12.2 1.22b kg b.i.d.) Ampicillin (1 mg/kg 1 c. +1d t.i.d.)-gentamicin (2. mg/kg b.i.d.) Controls.12 ±. a p <.1 when compared with all other groups. b p <.1 when compared with ampicillin and control groups. c After days of antibiotic therapy. d p>. when compared with controls sacrificed on postinfection day. icantly lower levels than those in untreated controls or those that received the vancomycin or ampicillin-gentamicin treatment regimens (Table 2). No vegetations were rendered culture negative by any of the treatment regimens. For animals infected with E. raffinosus SF-1, days of daptomycin, ampicillin-gentamicin, or vancomycin therapy significantly reduced intravegetation enterococcal densities compared with the densities in untreated controls and rabbits that underwent ampicillin monotherapy (Table ). Also, daptomycin, vancomycin, and ampicillin-gentamicin were equally effective at rendering vegetations culture negative, with rates ranging from to % (Table ), while ampicillin alone rendered few vegetations culture negative (%; P <. versus the other therapy groups). ISCUSSION In the past decade there has been an emergence of enterococcal strains that exhibit in vitro resistance to multiple 1-lactam antibiotics. This resistance has caused clinical problems, ranging from nosocomial colonization to severe, life-threatening infections, including endocarditis (,, 11, 1-1, 22). For,B-lactamase-producing, aminoglycoside-resistant E. faecalis, nosocomial spread has involved one of two modes: (i) a rapid and geographically confined clonal dissemination (1) or (ii) a slow and geographically dispersed spread over a number of hospital areas (22). The penicillinresistant, f-lactamase-negative enterococci, which have also been recently confirmed as important nosocomial pathogens (, 1, 21), have mainly been identified as E. faecium, E. raffinosus, and E. avium and are,-lactam resistant by virtue of the presence of penicillin-binding proteins with low binding affinities for these antibiotics (). Irrespective of the mechanism(s) of,b-lactam resistance, infections caused by such enterococcal strains present the clinician with limited therapeutic options. aptomycin is a new lipopeptide agent with significant in vitro activities against gram-positive cocci, including enterococci (, ). In the present study, we evaluated the in vivo activities of daptomycin against those of vancomycin and ampicillin (with and without gentamicin) in an animal model of,-lactam-resistant enterococcal infection (endocarditis). It provided a rigorous test of antibiotic efficacy. Several noteworthy observations emanated from this study. For the -lactamase-producing E. faecalis strain HH-22, daptomycin was significantly more effective in reducing intravegeta- ownloaded from http://aac.asm.org/ on November, 21 by guest
1 RAMOS ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE. aptomycin compared with vancomycin and ampicillin with or without gentamicin for therapy of experimental enterococcal endocarditis caused by E. raffinosus SF-1 No. Mean ± S log1o No. of culture-negative Regimen rabbits day CFU/g of vegetations/total no. of vegetation vegetations (%) aptomycin (2 mg/kg b.i.d.) 1 2.12 +.ab, 11/1 ()d Ampicillin (1 mg/kg t.i.d.)- 11 e 2.1 ±.1a,b /11 (2)d gentamicin (2. mg/kg b.i.d.) Vancomycin (2 mg/kg b.i.d.) 12 2.22 +.2a. /12 ()" Ampicillin (1 mg/kg t.i.d.) 1. + 1.a /1 () Controls.1 ± 1. / () a p <.1 versus control group. b p <.1 versus ampicillin-treatment group. c P >. versus vancomycin and ampicillin-gentamicin treatment groups. d p <. versus ampicillin monotherapy group. e After days of antibiotic therapy. tion enterococcal densities than were the other agents, including vancomycin and ampicillin-gentamicin. Thus, the presence of plasmid-mediated,-lactam-aminoglycoside resistance precludes the in vivo synergy of such combinations in vivo, and such strains are moderately susceptible to vancomycin. The data presented here regarding intermittent daptomycin administrations in the rabbit model confirm the effectiveness of this agent against P-lactamase-producing enterococci in vivo, as noted by others (). Other options for the management of enterococcal infections caused by P-lac- include combinations of P-lacta- tamase-producing strains mase inhibitors and f-lactams (1,, 1). In contrast, for E. raffinosus SF-1, both daptomycin and vancomycin were equally efficacious at reducing intravegetation bacterial densities; moreover, ampicillin alone was moderately effective at reducing intravegetation enterococcal densities (although few vegetations were rendered culture negative), while the addition of gentamicin to the ampicillin treatment rendered most vegetations culture negative, with mean vegetation bacterial densities being similar to those in daptomycin- or vancomycin-treated animals. Thus, enterococcal strains that are,-lactam resistant on the basis of low-affinity penicillinbinding proteins (e.g., PBP- in strain SF-1) may, in fact, respond to -lactam therapy, provided that the dose yields levels in serum in excess of the MIC. The current data also suggest that the levels of the 1-lactam (in this case, ampicillin) in serum were high enough to bind to the relevant penicillin-binding proteins in order to induce augmented aminoglycoside entry into the enterococcal cells (1). ata on treating human infections caused by -lactamresistant enterococci have been limited to date. Patterson et al. (1) described the cure of one patient with probable endocarditis caused by a,b-lactamase-producing, gentamicin-resistant strain with a -week course of vancomycin. In contrast, Wells et al. (22) noted the paradoxical cure of patients infected with -lactamase-producing, gentamicinresistant strains by seemingly inappropriate antibiotic regimens, while some patients who received apparently appropriate regimens were unsuccessfully treated. They ascribed the former outcome to a preponderance of urinary tract enterococcal infections and speculated that the poor treatment outcome in the latter situation was likely due to the severe debilitation suffered by a majority of these patients. In summary, the presence of,b-lactamase-producing, aminoglycoside-resistant enterococci in patients with infections should prompt the use of a non-1-lactam (e.g., vancomycin) or a P-lactam-1-lactamase inhibitor regimen; it seems likely that infections caused by,-lactamase-negative strains which are not highly resistant to -lactams () will respond to either,-lactam-aminoglycoside combinations or vancomycin. The good in vivo activities of vancomycin and daptomycin in both of our models of penicillin-resistant enterococcal endocarditis most likely reflect the unique cell wall sites of action of these agents (, 12) in comparison with the sites of action of P-lactam antibiotics. Although highly active in vitro and in vivo against the,b-lactam-resistant enterococci, daptomycin is not planned for further clinical development; congeners of this compound are being actively pursued (a). We anxiously await further reports describing the treatment of clinical enterococcal infections caused by,-lactam-resistant strains in order to define the optimal therapeutic options in such settings. ACKNOWLEGMENTS This study was supported in part by grants from the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil (FAPESP), and the Eli Lilly Research Laboratories, Indianapolis, Ind. REFERENCES 1. Bayer, A. S., and J. Tu. 1. Chemoprophylactic efficacy against experimental endocarditis caused by P-lactamase-producing, aminoglycoside-resistant enterococci is associated with prolonged serum inhibitory activity. Antimicrob. Agents Chemother. :1-1. 2. Bayer, A. S., J. Yih, and L. Hirano. 1. 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