Abstract
The activities of daptomycin, a cyclic lipopeptide, and eight other agents were determined against 338 strains of gram-positive anaerobic bacteria and corynebacteria by the NCCLS reference agar dilution method with supplemented brucella agar for the anaerobes and Mueller-Hinton agar for the corynebacteria. The daptomycin MICs determined on Ca2+-supplemented (50 mg/liter) brucella agar plates were one- to fourfold lower than those determined in unsupplemented media. Daptomycin was highly active (MICs, ≤2 μg/ml) against many strains including 36 of 37 peptostreptococci, 37 of 48 isolates of the Eubacterium group, and all strains of Propionibacterium spp., Clostridium perfringens, Clostridium difficile, and other Clostridium spp. It was fourfold or greater more active than vancomycin against Clostridium innocuum and 16 of 34 strains of vancomycin-resistant lactobacilli. Three strains of C. difficile for which quinupristin-dalfopristin and linezolid MICs were >8 μg/ml were inhibited by <1 μg of daptomycin per ml. Daptomycin MICs were ≥4 μg/ml for most strains of Clostridium clostridioforme, Clostridium paraputrificum, Clostridium tertium, and Clostridium ramosum; the isolates were generally more resistant to other antimicrobials. Daptomycin was two- to fourfold less active against Actinomyces spp. than vancomycin, quinupristin-dalfopristin, or linezolid. Twenty-nine of 31 strains of Corynebacterium spp., including Corynebacterium jeikeium, Corynebacterium amycolatum, and Corynebacterium pseudodiphtheriticum, were inhibited by ≤0.25 μg of daptomycin per ml. For two strains of “Corynebacterium aquaticum,” 8 μg of daptomycin per ml was required for inhibition. Daptomycin demonstrated very good activities against a broad range of gram-positive organisms including vancomycin-resistant C. innocuum and lactobacillus strains and quinupristin-dalfopristin- and linezolid-resistant C. difficile strains.
Daptomycin is a cyclic lipopeptide antibiotic derived from Streptomyces roseoporus with bactericidal activity against resistant gram-positive bacteria including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and penicillin-resistant pneumococci. It is undergoing clinical trials at present (24). Daptomycin acts by disrupting the bacterial cytoplasmic membrane and by dissipation of the membrane potential (1). Spontaneous resistance to daptomycin is rare (22).
A few studies performed in the late 1980s or early 1990s have noted that daptomycin is active against a very limited number of anaerobes (4, 6, 9, 13, 21). Since the anaerobic gram-positive isolates involved in diverse clinical infections comprise a large variety of species and many clinical laboratories are either unable to perform or choose not to perform anaerobic susceptibility studies, clinicians are often reliant on the published literature to select both empirical and specific therapies. Because of the unusual resistance patterns recently manifested by gram-positive anaerobes such as Clostridium and Peptostreptococcus species (2, 5, 22), information on recent isolates is desirable. In addition, most of the prior studies of the activity of daptomycin against anaerobes used Wilkins-Chalgren agar or brain heart infusion agar (6, 9, 11, 13, 15), but these have now been replaced by supplemented brucella blood agar as the current NCCLS anaerobic reference susceptibility testing medium (18). Therefore, using more contemporary isolates, a larger number of defined species, and supplemented brucella blood agar, we evaluated the potential utility of daptomycin against a variety of recently isolated anaerobic gram-positive organisms. A set of pathogenic corynebacterial strains was tested under aerobic conditions by NCCLS methods (17).
The antibacterial activity of daptomycin against aerobes has been noted to be affected by the free Ca2+ concentration, requiring supplementation of the growth medium to achieve the levels encountered in normal human serum (∼50 mg/liter) (3, 14, 24). To determine the effect of Ca2+ supplementation on daptomycin MICs for anaerobes, we compared non-calcium-supplemented with calcium-supplemented brucella agar.
MATERIALS AND METHODS
Isolates from human clinical specimens were identified by standard criteria (2, 16, 23). Strains of anaerobic gram-positive anaerobes and corynebacteria were taken from a freezer and subcultured at least twice on brucella agar supplemented with vitamin K1, hemin, and laked sheep blood or Trypticase soy blood agar, respectively, to ensure purity and good growth. The species and numbers of isolates tested are listed in Table 1. Control strains included Staphylococcus aureus ATCC 29213 and Eubacterium lentum ATCC 40335. Agar dilution tests were performed according to NCCLS guidelines (17, 18). Antimicrobial agents were reconstituted according to the instructions of the manufacturers, serially diluted, and added to molten supplemented brucella agar for the anaerobes and to Mueller-Hinton agar for the corynebacteria. Daptomycin plates were prepared with and without additional Ca2+ to achieve a daptomycin concentration of 50 mg/liter. The calcium concentrations in the agar and broth media were measured with a Beckman Synchron CX 7 analyzer to determine the amount of supplementation needed to achieve a concentration of 50 mg/liter. Drug-free plates were inoculated before and after inoculation of each antimicrobial plate series and were used as growth controls.
TABLE 1.
In vitro activities of daptomycin and seven other antimicrobial agents against gram-positive anaerobes and corynebacteria
| Organism (no. of isolates) and antimicrobial agent | MIC (μg/ml)
|
||
|---|---|---|---|
| Range | 50% | 90% | |
| Actinomyces group (22)a | |||
| Daptomycin + calcium (50 mg/liter) | 0.06-16 | 2 | 4 |
| Daptomycin | 0.25->32 | 8 | 32 |
| Quinupristin-dalfopristin | 0.25-1 | 0.5 | 0.5 |
| Synercid | 0.06-0.5 | 0.125 | 0.25 |
| Vancomycin | 0.5-1 | 0.5 | 1 |
| Ampicillin | ≤0.03-0.5 | 0.06 | 0.25 |
| Penicillin | ≤0.03-0.5 | 0.06 | 0.25 |
| Imipenem | ≤0.03-0.25 | 0.06 | 0.125 |
| Piperacillin-tazobactam | ≤0.03-1 | 0.25 | 1 |
| Bifidobacterium spp. (13)b | |||
| Daptomycin + calcium (50 mg/liter) | ≤0.03-1 | 0.25 | 0.5 |
| Daptomycin | 0.25-4 | 0.5 | 4 |
| Quinupristin-dalfopristin | 0.25-2 | 1 | 1 |
| Synercid | 0.125-0.5 | 0.25 | 0.25 |
| Vancomycin | 0.25-1 | 0.5 | 1 |
| Ampicillin | ≤0.03-1 | 0.125 | 0.5 |
| Penicillin | ≤0.03-0.5 | 0.125 | 0.5 |
| Imipenem | ≤0.03-0.25 | 0.06 | 0.06 |
| Piperacillin-tazobactam | ≤0.03-1 | 0.06 | 0.5 |
| Clostridium bifermentans-Clostridium sordellii group (10)c | |||
| Daptomycin + calcium (50 mg/liter) | 0.5-8 | 2 | 4 |
| Daptomycin | 2-16 | 8 | 16 |
| Quinupristin-dalfopristin | 1-1 | 1 | 1 |
| Synercid | 0.25-1 | 0.5 | 0.5 |
| Vancomycin | 0.25-1 | 0.5 | 1 |
| Ampicillin | ≤0.03-0.5 | 0.06 | 0.5 |
| Penicillin | ≤0.03-0.25 | ≤0.03 | 0.25 |
| Imipenem | ≤0.03-0.5 | 0.06 | 0.5 |
| Piperacillin-tazobactam | ≤0.03-1 | 0.125 | 0.5 |
| Clostridium cadaveris (10) | |||
| Daptomycin + calcium (50 mg/liter) | 0.5-8 | 2 | 4 |
| Daptomycin | 1->32 | 4 | 16 |
| Quinupristin-dalfopristin | 2-4 | 4 | 4 |
| Synercid | 0.25-1 | 0.5 | 1 |
| Vancomycin | 1-4 | 2 | 2 |
| Ampicillin | ≤0.03-1 | 0.125 | 1 |
| Penicillin | 0.06-1 | 0.125 | 1 |
| Imipenem | 0.06-0.5 | 0.125 | 0.25 |
| Piperacillin-tazobactam | ≤0.03-2 | 0.06 | 0.5 |
| Clostridium clostridioforme (10) | |||
| Daptomycin + calcium (50 mg/liter) | 0.5-8 | 2 | 4 |
| Daptomycin | 0.25-8 | 2 | 4 |
| Quinupristin-dalfopristin | 2-4 | 2 | 4 |
| Synercid | 0.5-8 | 2 | 8 |
| Vancomycin | 0.25-1 | 0.5 | 1 |
| Ampicillin | 0.5->128 | 1 | 2 |
| Penicillin | 0.25->32 | 0.5 | 2 |
| Imipenem | 0.5-2 | 1 | 2 |
| Piperacillin-tazobactam | 0.125->32 | 1 | 8 |
| Clostridium difficile (18) | |||
| Daptomycin + calcium (50 mg/liter) | 0.125-1 | 0.5 | 1 |
| Daptomycin | 0.5-4 | 1 | 2 |
| Quinupristin-dalfopristin | 2-16 | 2 | 16 |
| Synercid | 0.5-16 | 1 | 16 |
| Vancomycin | 0.5-4 | 1 | 2 |
| Ampicillin | 2-4 | 2 | 4 |
| Penicillin | 1-4 | 2 | 4 |
| Imipenem | 4-16 | 8 | 16 |
| Piperacillin-tazobactam | 4-16 | 8 | 16 |
| Clostridium innocuum (19) | |||
| Daptomycin + calcium (50 mg/liter) | 1-4 | 2 | 4 |
| Daptomycin | 4-16 | 8 | 16 |
| Quinupristin-dalfopristin | 2-4 | 4 | 4 |
| Synercid | 0.5-4 | 1 | 2 |
| Vancomycin | 8-32 | 16 | 16 |
| Ampicillin | 0.06-0.25 | 0.25 | 0.25 |
| Penicillin | 0.125-0.5 | 0.25 | 0.5 |
| Imipenem | 1-4 | 2 | 4 |
| Piperacillin-tazobactam | 0.5-2 | 1 | 1 |
| Clostridium paraputrificum-Clostridium tertium group (10)d | |||
| Daptomycin + calcium (50 mg/liter) | 2-32 | 8 | 32 |
| Daptomycin | 16->32 | >32 | >32 |
| Quinupristin-dalfopristin | 1-8 | 4 | 4 |
| Synercid | 0.25-1 | 1 | 1 |
| Vancomycin | 0.5-2 | 1 | 2 |
| Ampicillin | 0.06-2 | 0.5 | 1 |
| Penicillin | 0.06-2 | 0.125 | 1 |
| Imipenem | 0.125-0.5 | 0.25 | 0.5 |
| Piperacillin-tazobactam | 0.125-32 | 0.5 | 16 |
| Clostridium perfringens (11) | |||
| Daptomycin + calcium (50 mg/liter) | 0.06-0.5 | 0.5 | 0.5 |
| Daptomycin | 0.125-4 | 2 | 4 |
| Quinupristin-dalfopristin | 1-4 | 2 | 2 |
| Synercid | 0.5-1 | 1 | 1 |
| Vancomycin | 0.5-0.5 | 0.5 | 0.5 |
| Ampicillin | ≤0.03-0.06 | ≤0.03 | 0.06 |
| Penicillin | ≤0.03-0.125 | 0.06 | 0.06 |
| Imipenem | 0.06-0.125 | 0.125 | 0.125 |
| Piperacillin-tazobactam | ≤0.03-0.25 | ≤0.03 | 0.25 |
| Clostridium ramosum (15) | |||
| Daptomycin + calcium (50 mg/liter) | 1-16 | 16 | 16 |
| Daptomycin | 8->32 | 32 | >32 |
| Quinupristin-dalfopristin | 4-8 | 8 | 8 |
| Synercid | 0.25-4 | 1 | 4 |
| Vancomycin | 2-8 | 8 | 8 |
| Ampicillin | 0.06-0.5 | 0.125 | 0.25 |
| Penicillin | ≤0.03-2 | 0.125 | 2 |
| Imipenem | 0.125-0.25 | 0.25 | 0.25 |
| Piperacillin-tazobactam | 0.06-2 | 0.06 | 1 |
| Clostridium spp. (25)e | |||
| Daptomycin + calcium (50 mg/liter) | ≤0.03-2 | 0.5 | 2 |
| Daptomycin | ≤0.03-8 | 2 | 8 |
| Quinupristin-dalfopristin | 0.25-4 | 1 | 4 |
| Synercid | 0.125-8 | 0.5 | 1 |
| Vancomycin | ≤0.06-8 | 1 | 2 |
| Ampicillin | ≤0.03->128 | 0.25 | 0.5 |
| Penicillin | ≤0.03->32 | 0.125 | 0.25 |
| Imipenem | ≤0.03-1 | 0.125 | 0.5 |
| Piperacillin-tazobactam | ≤0.03-2 | 0.25 | 2 |
| Eubacterium lentum (17) | |||
| Daptomycin + calcium (50 mg/liter) | 0.125-16 | 2 | 16 |
| Daptomycin | 1->32 | 8 | >32 |
| Quinupristin-dalfopristin | 1-2 | 1 | 2 |
| Synercid | 0.25-2 | 0.5 | 1 |
| Vancomycin | 0.5-2 | 2 | 2 |
| Ampicillin | 0.25-2 | 0.5 | 2 |
| Penicillin | ≤0.03-2 | 1 | 2 |
| Imipenem | ≤0.03-0.5 | 0.25 | 0.5 |
| Piperacillin-tazobactam | ≤0.03-32 | 16 | 32 |
| Eubacterium group spp. (31)f | |||
| Daptomycin + calcium (50 mg/liter) | ≤0.03-16 | 0.25 | 4 |
| Daptomycin | 0.06->32 | 1 | 16 |
| Quinupristin-dalfopristin | 0.06-8 | 1 | 8 |
| Synercid | ≤0.03-4 | 0.5 | 1 |
| Vancomycin | 0.25-2 | 0.5 | 2 |
| Ampicillin | ≤0.03-1 | 0.125 | 0.25 |
| Penicillin | ≤0.03-2 | 0.125 | 2 |
| Imipenem | ≤0.03-0.25 | ≤0.03 | 0.06 |
| Piperacillin-tazobactam | ≤0.03-16 | 0.06 | 0.5 |
| Lactobacillus spp. (37)g | |||
| Daptomycin + calcium (50 mg/liter) | ≤0.03-32 | 1 | 16 |
| Daptomycin | 0.125->32 | 4 | >32 |
| Quinupristin-dalfopristin | 0.5-16 | 4 | 8 |
| Synercid | 0.125-4 | 1 | 2 |
| Vancomycin | 0.25->32 | 4 | >32 |
| Ampicillin | ≤0.03-8 | 0.5 | 2 |
| Penicillin | ≤0.03-4 | 0.5 | 2 |
| Imipenem | ≤0.03-8 | 0.125 | 2 |
| Piperacillin-tazobactam | ≤0.03-8 | 1 | 2 |
| Peptostreptococcus asaccharolyticus (10) | |||
| Daptomycin + calcium (50 mg/liter) | ≤0.03-0.125 | ≤0.03 | 0.06 |
| Daptomycin | ≤0.03-0.5 | 0.06 | 0.125 |
| Quinupristin-dalfopristin | 0.5-1 | 1 | 1 |
| Synercid | 0.25-1 | 0.5 | 1 |
| Vancomycin | 0.125-0.5 | 0.125 | 0.25 |
| Ampicillin | ≤0.03-1 | ≤0.03 | 0.25 |
| Penicillin | ≤0.03-0.5 | ≤0.03 | 0.25 |
| Imipenem | ≤0.03-0.06 | ≤0.03 | ≤0.03 |
| Piperacillin-tazobactam | ≤0.03-1 | ≤0.03 | 0.25 |
| Peptostreptococcus magnus- Peptostreptococcus micros group (14)h | |||
| Daptomycin + calcium (50 mg/liter) | 0.125-1 | 0.5 | 1 |
| Daptomycin | 1-4 | 2 | 4 |
| Quinupristin-dalfopristin | 0.5-4 | 1 | 2 |
| Synercid | 0.25-2 | 1 | 2 |
| Vancomycin | 0.25-1 | 0.5 | 1 |
| Ampicillin | 0.06-1 | 0.25 | 1 |
| Penicillin | ≤0.03-0.25 | ≤0.03 | 0.25 |
| Imipenem | ≤0.03-0.125 | ≤0.03 | 0.125 |
| Piperacillin-tazobactam | ≤0.03-0.5 | ≤0.03 | 0.25 |
| Peptostreptococcus spp. (13)i | |||
| Daptomycin + calcium (50 mg/liter) | ≤0.03-16 | 0.25 | 2 |
| Daptomycin | ≤0.03->32 | 1 | 16 |
| Quinupristin-dalfopristin | 0.5-16 | 1 | 2 |
| Synercid | 0.125-1 | 0.25 | 0.5 |
| Vancomycin | 0.125-1 | 0.5 | 0.5 |
| Ampicillin | ≤0.03-4 | 0.125 | 0.25 |
| Penicillin | ≤0.03-2 | 0.06 | 0.25 |
| Imipenem | ≤0.03-0.5 | ≤0.03 | 0.125 |
| Piperacillin-tazobactam | ≤0.03-8 | 0.25 | 1 |
| Propionibacterium spp. (15)j | |||
| Daptomycin + calcium (50 mg/liter) | 0.125-2 | 0.5 | 2 |
| Daptomycin | 0.5-8 | 4 | 8 |
| Quinupristin-dalfopristin | 0.25-1 | 0.5 | 1 |
| Synercid | 0.125-0.25 | 0.125 | 0.25 |
| Vancomycin | 0.5-1 | 0.5 | 0.5 |
| Ampicillin | ≤0.03-0.25 | 0.125 | 0.25 |
| Penicillin | ≤0.030-0.06 | 0.06 | 0.06 |
| Imipenem | ≤0.03-≤0.03 | ≤0.03 | ≤0.03 |
| Piperacillin-tazobactam | ≤0.03-1 | ≤0.03 | 1 |
| Corynebacterium jeikeium (10) | |||
| Daptomycin + calcium (50 mg/liter) | 0.125-0.5 | 0.25 | 0.25 |
| Daptomycin | 1-4 | 1 | 2 |
| Quinupristin-dalfopristin | 0.5-0.5 | 0.5 | 0.5 |
| Synercid | 0.25-1 | 0.25 | 0.25 |
| Vancomycin | 0.25-0.5 | 0.5 | 0.5 |
| Ampicillin | 128->128 | >128 | >128 |
| Penicillin | >32->32 | >32 | >32 |
| Imipenem | 4->32 | >32 | >32 |
| Piperacillin-tazobactam | >32->32 | >32 | >32 |
| Corynebacterium spp. (21)k | |||
| Daptomycin + calcium (50 mg/liter) | ≤0.03-8 | ≤0.03 | 1 |
| Daptomycin | ≤0.03->32 | 0.25 | 8 |
| Quinupristin-dalfopristin | 0.25-1 | 0.5 | 1 |
| Synercid | 0.06-2 | 0.25 | 1 |
| Vancomycin | ≤0.06-2 | 0.25 | 0.5 |
| Ampicillin | ≤0.03-64 | 0.5 | 4 |
| Penicillin | ≤0.03->32 | 0.5 | 16 |
| Imipenem | ≤0.03->32 | ≤0.03 | 2 |
| Piperacillin-tazobactam | ≤0.03-32 | 2 | 32 |
A. israelii (n = 4), A. meyeri (n = 4), A. naeslundii (n = 4), A. odontolyticus (n = 5), A. viscosus (n = 4), and Arcanobacterium pyogenes (n = 1).
B. adolescentis (n = 2), B. bifidum (n = 1), B. breve (n = 2), B. catenulatum (n = 2), B. dentium (n = 1), B. longum (n = 1), and no good fit (n = 4).
C. bifermentans (n = 5) and C. sordellii (n = 5).
C. paraputrificum (n = 5) and C. tertium (n = 5).
C. aminovalericum (n = 1), C. baratii (n = 1), C. butyricum (n = 5), C. cochlearium (n = 1), C. glycolicum (n = 2), C. leptum (n = 1), C. novyi A (n = 1), C. sphenoides (n = 1), C. sporogenes (n = 1), C. subterminale (n = 3), C. symbiosum (n = 5), and no good fit (n = 3).
Collinsella aerofaciens (n = 9), E. alactolyticum (n = 1), E. brachy (n = 1), E. combesii (n = 1), E. contortum (n = 2), E. limosum (n = 6), and no good fit (n = 11).
L. acidophilus (n = 3), L. brevis (n = 2), L. casei (n = 7), L. catenaforme (n = 2), L. contusus (n = 1), L. delbrueckii (n = 1), L. fermentum (n = 1), L. jensenii (n = 1), L. lactis (n = 1), L. plantarum (n = 2), L. rhamnosus (n = 1), and no good fit (n = 15).
P. magnus (n = 7) and P. micros (n = 7).
Gemella morbillorum (n = 1), P. anaerobius (n = 9), and P. prevotii (n = 3).
P. acnes (n = 5), P. avidum (n = 7) and P. granulosum (n = 3).
C. afermentans (n = 1), C. amycolatum (n = 6), C. aquaticum (n = 3), C. minutissimum (n = 1), C. pseudodiphtheriticum (n = 4), C. striatum (n = 2), C. urealyticum (n = 1), C. xerosis (n = 1), no good fit (n = 1), and Corynebacterium group F (n = 1).
Colonies were suspended in brucella broth to a turbidity equal to that of a 0.5 McFarland standard and applied to the plates with a Steers replicator that delivered approximately 105 CFU/spot for anaerobes. The anaerobic plates were incubated in the anaerobic chamber incubator for 44 h at 36°C prior to interpretation of the results. Corynebacteria were tested with an inoculum of 104 CFU/spot, and the plates were incubated at 36°C for 24 h under ambient conditions. The MIC was the concentration that completely inhibited growth or caused a marked reduction in the appearance of growth compared to that for the drug-free growth control (17, 18).
RESULTS
The results of our in vitro study are presented in Table 1. The modal daptomycin MIC for the S. aureus ATCC 29213 control organisms on calcium-supplemented plates was 0.5 μg/ml, while the modal MIC on unsupplemented agar was 2 μg/ml, demonstrating that calcium supplementation is required for agar dilution tests. The MICs for the anaerobic isolates tested on brucella agar plates supplemented with calcium were generally one- to fourfold lower than those on unsupplemented plates.
Daptomycin was highly active against many strains. The MICs for 18 strains of Clostridium difficile were ≤1 μg/ml; these included 3 strains for which linezolid MICs were 8 to 16 μg/ml, 3 strains for which quinupristin-dalfopristin MICs were 8 to 16 μg/ml, and 3 strains for which imipenem MICs were 16 μg/ml. Daptomycin was also active (MICs, 1 to 4 μg/ml) against 19 strains of Clostridium innocuum for which vancomycin MICs were 8 to 32 μg/ml. All strains of Clostridium perfringens were inhibited by 0.06 to 0.5 μg of daptomycin per ml. Daptomycin MICs were ≥4 μg/ml for most strains of Clostridium ramosum, Clostridium paraputrificum, and Clostridium tertium; and these strains were generally more resistant to the other agents tested. The daptomycin MIC for Clostridium clostridioforme, one of the clostridia that stains gram negative, did not demonstrate the shift to a lower value in the Ca2+-supplemented medium.
Daptomycin showed excellent activity (MIC, ≤1 μg/ml) against all peptostreptococci except one strain of Peptostreptococcus prevotii. Members of the Eubacterium group were generally susceptible, with MICs only occasionally being >4 μg/ml for strains of E. lentum and Collinsella aerofaciens. All strains of Propionibacterium spp. were susceptible to ≤2 μg of daptomycin per ml and were also susceptible to the other agents tested. Daptomycin MICs were ≥16 μg/ml for 5 of 32 strains of Lactobacillus spp., but 16 vancomycin-resistant strains were all susceptible to daptomycin. Vancomycin was generally two- to fourfold more active than daptomycin against Actinomyces spp.
Penicillin and the other beta-lactam agents tested were the most active agents tested against Actinomyces group species and many of the clostridia including Clostridium bifermentans, Clostridium sordellii, C. clostridioforme, C. innocuum, and C. perfringens. The daptomycin MICs were within 1 or 2 dilutions higher than the vancomycin MICs for the clostridia. Daptomycin was fourfold or greater more active than vancomycin against C. innocuum. The activities of quinupristin-dalfopristin and linezolid were more variable than those of daptomycin against the species tested.
DISCUSSION
Daptomycin is undergoing clinical evaluation for the treatment of infections caused by drug-resistant gram-positive organisms (24). While most attention has focused on infections caused by methicillin-resistant S. aureus and drug-resistant enterococcal and pneumococcal strains, daptomycin may potentially be used against anaerobes as well. Jones and Barry (15) and Fuchs et al. (10) proposed tentative interpretive criteria of a daptomycin MIC of ≤2 μg/ml for susceptible strains and a daptomycin MIC of ≥8 μg/ml for resistant strains. Calcium supplementation of brucella-based media resulted in MICs one- to fourfold lower on plates supplemented with Ca2+ (50 mg/liter) than on plates not supplemented with calcium. This is in accord with the findings of others for anaerobes with Wilkins-Chalgren agar and brain heart infusion agar (6, 9, 11, 13, 15). Similar results were noted in our study with the corynebacteria tested, as previously noted by others (3, 8, 10) for aerobes, also with Mueller-Hinton medium. Supplementation with calcium to physiologic levels appears to be required for accurate daptomycin MIC determinations by both aerobic and anaerobic agar dilution testing, regardless of the basal medium used.
Clinical failures of metronidazole therapy of C. difficile-associated colitis and the in vitro resistance of C. difficile isolates to metronidazole have been reported (5). While vancomycin has been the alternative agent of choice for such cases, the development of vancomycin-resistant enterococci has led to guidelines suggesting “the prudent use of vancomycin” (12). The development of alternative agents for the treatment of infections caused by such organisms seems desirable. Daptomycin has been noted to be active in vitro against C. difficile (4, 9, 13, 21). Faruki et al. (9), using Wilkins-Chalgren broth, studied 62 C. difficile isolates and found an MIC at which 90% of isolates are inhibited (MIC90) of 0.12 μg/ml with calcium-supplemented Wilkins-Chalgren agar and an MIC90 of 8 μg/ml with unsupplemented agar. Bartoloni et al. (4) found a daptomycin MIC90 of 0.87 μg/ml and suggested that it might be effective as therapy for C. difficile-associated colitis. This was supported by the study of Silva et al. (21), who used supplemented brain heart infusion agar and who noted that daptomycin is more active in vitro than both metronidazole and vancomycin against C. difficile isolates. In our study daptomycin was more active than vancomycin, linezolid, or quinupristin-dalfopristin against C. difficile isolates. Three strains of C. difficile for which quinupristin-dalfopristin and linezolid MICs were >8 μg/ml were inhibited by daptomycin at <1 μg/ml.
Dong et al. (7) reported that daptomycin at 0.05 mg/day was effective in preventing death in a hamster C. difficile colitis model. Nord et al. (19) studied the in vitro activity of daptomycin against C. difficile and subsequently (20) used 5 mg/day and also found that it was protective in the hamster model. At present, daptomycin therapy involves intravenous dosing; an oral formulation would be needed to treat C. difficile-associated diarrheal diseases.
Greenwood and Palfreyman (11) reported that daptomycin is active against Peptostreptococcus species, but they noted a dramatic potentiation of MICs related to calcium supplementation of the growth medium. They noted a wide range of MICs (≤0.12 to >8 μg/ml) but did not identify the species of their isolates. Silva et al. (21) studied the activities of daptomycin against 23 peptostreptococcal strains (the species were not identified) and found that a daptomycin concentration of >4 μg/ml was required for inhibition of 26% of the isolates, but they noted a broad range of MICs (0.5 to 32 μg/ml). Our study noted variations in susceptibility to daptomycin by species. All Peptostreptococcus asaccharolyticus isolates were susceptible to daptomycin at ≤0.125 μg/ml, all Peptostreptococcus micros strains were susceptible to daptomycin at ≤0.5 μg/ml, and Peptostreptococcus magnus isolates required up to 1 μg of daptomycin per ml for inhibition, while one of three Peptostreptococcus prevotii strains required 16 μg of daptomycin per ml for inhibition.
Barry et al. (3) studied 10 Lactobacillus isolates, many of which were resistant to vancomycin, and found an MIC90 of 4.0 μg/ml on calcium-supplemented medium and an MIC90 of 8.0 μg/ml on unsupplemented medium. Our study noted a bimodal pattern of susceptibility of lactobacilli to daptomycin. While most strains were susceptible to daptomycin at ≤2 μg/ml, two strains of Lactobacillus plantarum, one strain of Lactobacillus rhamnosus, and one strain of Lactobacillus whose species was unknown but that was also metronidazole resistant required >16 μg of daptomycin per ml for inhibition. Daptomycin was fourfold or greater more active than vancomycin against 16 of 34 strains of vancomycin-resistant lactobacilli.
Our study showed that daptomycin inhibited 29 of 31 strains of Corynebacterium spp. at ≤0.25 μg/ml, including 10 strains of Corynebacterium jeikeium that were resistant to penicillin, ampicillin, imipenem, and piperacillin-tazobactam. Two strains of Corynebacterium aquaticum required treatment with 8 μg/ml for inhibition. Barry et al. (3) reported the activities of daptomycin against 22 Corynebacterium sp. isolates (the species were not identified) and 7 C. jeikeium strains and reported an MIC90 of 0.5 μg/ml on calcium-supplemented medium and an MIC90 of 1.0 μg/ml on unsupplemented medium, but they did not report any variation in MIC by species. Fuchs et al. (10) noted that the geometric mean MIC of daptomycin for 21 isolates of C. jeikeium was 0.25 μg/ml and that it is more active than vancomycin and teichoplanin. Eliopoulos et al. (8) studied unidentified diphtheroids and noted that the daptomycin MIC90 for the isolates was 4 μg/ml.
Daptomycin demonstrated very good activities against a broad range of anaerobic gram-positive organisms including vancomycin-resistant C. innocuum and lactobacillus strains and quinupristin-dalfopristin-resistant strains of C. difficile. Due to marked differences in the patterns of susceptibility to daptomycin by species, laboratories should consider identification of the species of clinically significant anaerobic isolates, especially when susceptibility studies are not routinely performed.
Acknowledgments
This study was funded, in part, by a grant from Cubist Pharmaceuticals, Lexington, Mass.
We thank Judee H. Knight and Alice E. Goldstein for assistance.
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