Activity of Daptomycin against Recent North American Isolates of Streptococcus pneumoniae

Abstract

Daptomycin MICs at which 50% of isolates were inhibited (MIC₅₀s) and MIC₉₀s determined by the NCCLS broth microdilution method were both 0.25 μg/ml (range, 0.06 to 2 μg/ml) for 350 pneumococcal isolates. MICs determined by E test strips on commercially prepared Mueller-Hinton sheep blood agars with different calcium contents were 2 to 3 dilutions higher than those determined by strips that contained daptomycin plus calcium. Daptomycin zone diameters varied little on the same media.

The increasing resistance of Streptococcus pneumoniae over the past decade to several different antibiotic classes has limited the therapeutic choices for treatment of severe, invasive pneumococcal infections (5, 8, 9, 25, 28, 29). Macrolide resistance has increased steadily in the United States (16, 29), with some resulting clinical failures (18; D. M. Musher, M. E. Dowell, V. D. Shortridge, R. K. Flamm, J. H. Jorgensen, P. Le Magueres, and K. L. Krause, Letter, N. Engl. J. Med. 346:630-631, 2002). The emergence of fluoroquinolone resistance in some areas has raised concern for treatment of serious infections in the future (6, 14, 15) and has also resulted in therapeutic failures (7, 15). In particular, fluoroquinolones have been recommended as agents with predictable activity against otherwise-drug-resistant pneumococci, especially for patients with underlying risk factors for severe infections (4, 13). The use of vancomycin to treat pneumococcal infections other than meningitis has not been encouraged, in part due to concerns about increasing vancomycin resistance in other organisms (13). Thus, newer agents that can provide predictable activity against multidrug-resistant pneumococcal strains in severely ill patients are needed.

Daptomycin is a semisynthetic lipopeptide antibiotic that appears to possess a unique mechanism of action: it disrupts the cell membranes of gram-positive bacteria by causing the loss of membrane potential (1). Because of very good reported activity against a variety of gram-positive bacteria both recently (3, 11, 23, 26) and when undergoing initial development (2, 17, 24), daptomycin is currently under development for several clinical indications and is administered once daily by the intravenous route (27). The optimal activity of daptomycin requires physiological levels of calcium ions (∼50 μg/ml), including levels in culture media used to perform susceptibility tests (3, 10, 12). This in vitro study assessed the activity of daptomycin against 350 recent North American surveillance isolates of S. pneumoniae, including selected highly resistant strains. In addition, selected isolates were examined by the E test gradient diffusion method and the NCCLS disk diffusion procedure for determination of daptomycin susceptibilities.

The 350 pneumococcal isolates examined in this study included 200 with resistance mechanisms affecting macrolides, lincosamides, streptogramins, fluoroquinolones, and beta-lactams. An additional 150 fully susceptible isolates were selected randomly from among more than 9,000 invasive pneumococcal isolates recovered and characterized during the Centers for Disease Control and Prevention's Active Bacterial Core Surveillance, part of the Emerging Infections Program, conducted from 1998 through 2000. The isolates were recovered from patients in seven different surveillance sites, which represented a total survey population of greater than 17 million people. These strains had been tested earlier against a battery of 15 antimicrobial agents representing all clinically relevant drug classes. The statistical power of the sample allowed detection of a resistance rate of approximately 0.2% (95% confidence interval, 0 to 1% resistance).

In addition to being tested with daptomycin, each isolate was tested with linezolid, quinupristin-dalfopristin, penicillin, cefotaxime, erythromycin, clindamycin, and levofloxacin. Daptomycin reagent grade powder was kindly provided by Cubist Pharmaceuticals. Diagnostic grade powders of the other agents were either obtained from their manufacturers or purchased from Sigma Chemical Company, St. Louis, Mo. The test medium for the NCCLS broth microdilution procedure (20) was cation-adjusted Mueller-Hinton broth (Difco formulation; Becton Dickinson [BD], Cockeysville, Md.) supplemented with 3% lysed horse blood and, in addition, supplemented to contain a final calcium concentration of 44 μg/ml. Inocula of the test organisms were prepared from colonies grown on sheep blood agar plates incubated for 20 to 24 h in 5% CO₂. Colonies were suspended in 0.9% saline to obtain a suspension equivalent to the turbidity of a 0.5 McFarland standard and further diluted 1:10 in 0.9% saline within 15 min. This provided a final inoculum density of approximately 5 × 10⁵ CFU/ml in the wells of the microdilution panels following inoculation. Colony counts of positive control wells were performed periodically to verify the desired inoculum concentrations. The microdilution panels were incubated at 35°C in ambient air for 20 to 24 h prior to visual determination of MICs.

MICs for a subset of 50 selected strains were determined with specially prepared daptomycin E strips (prepared by AB Biodisk, Solna, Sweden, and provided by Cubist), one lot of which contained added calcium as well as the daptomycin to establish a calcium and daptomycin concentration gradient in the agar. The daptomycin concentration range for both E test formulations was 0.016 to 256 μg/ml. E test strips were applied to the surfaces of 150-mm-diameter Mueller-Hinton sheep blood agar plates (purchased from BD or Remel, Lenexa, Kans.). E strips containing vancomycin as a control drug were included on each plate. Plates were inoculated with a 0.5 McFarland density organism suspension prepared in 0.9% saline as described above. Plates were incubated at 35°C in 5% CO₂ for 20 to 24 h prior to determination of MICs. The MIC was defined by the intersection of the growth ellipse margin with the E test strip when reflected light at an oblique angle was used to examine the plates. Because the E test strips are marked in 1/2 log₂ concentration values, it was possible to record MICs in smaller than the usual twofold increments. In such instances in this study, E test MICs were rounded to the next higher log₂ MICs for purposes of comparison with the reference broth microdilution MICs.

Disk diffusion tests with 30-μg daptomycin disks (prepared by BD and provided by Cubist) were performed on the same 50 selected isolates according to the methods recommended by the NCCLS (21) with the same 150-mm Mueller-Hinton sheep blood agar plates (BD and Remel). Chemical assays of the calcium levels of the Mueller-Hinton agar lots used in the study were kindly provided by Cubist. Disk tests with vancomycin as the control drug were performed on each isolate. Plates were inoculated with an organism suspension equivalent to a 0.5 McFarland standard prepared in 0.9% saline as described above. Plates were incubated at 35°C in 5% CO₂ for 20 to 24 h prior to measurement of zone diameters.

S. pneumoniae ATCC 49619 was tested initially and with each day's MIC and disk diffusion tests for quality control purposes. MICs and zone diameter values for the control drug, vancomycin, were always within the NCCLS-specified range (22) throughout the study.

The daptomycin MIC at which 50% of the isolates were inhibited (MIC₅₀) and MIC₉₀ were both 0.25 μg/ml for the overall strain collection based on MICs determined by the NCCLS broth microdilution test method and calcium-supplemented lysed horse blood broth (Table 1). Linezolid was active against all strains tested, although three isolates were resistant to quinupristin-dalfopristin and 32 isolates were resistant to levofloxacin (Table 1). The daptomycin MICs for the resistant strain collection were slightly higher than those for the fully susceptible strains for unclear reasons.

TABLE 1.

Susceptibility of the S. pneumoniae isolate collection to daptomycin and the comparative agents by the NCCLS broth microdilution method with calcium-supplemented Mueller-Hinton lysed horse blood broth as the test medium

Drug and set of isolates	MIC (μg/ml)			% Resistant
	50%	90%	Range
All 350 isolates
Daptomycin	0.25	0.25	0.06-2	NAb
Linezolid	2	2	0.25-2	0
Quinupristin-dalfopristin	0.25	0.5	0.12-8	0.8
200 selected drug-resistant strains
Daptomycin	0.25	0.25	0.06-2	NA
Linezolid	2	2	0.25-2	0
Quinupristin-dalfopristin	0.25	0.5	0.12-8	1.5
Penicillin	1	4	≤0.03-8	46
Cefotaximea	1	4	≤0.03-16	36.5
Erythromycin	4	>64	≤0.03->64	60
Clindamycin	0.12	>32	≤0.03->32	25
Levofloxacin	1	16	0.5-32	16
150 selected drug-susceptible strains
Daptomycin	0.25	0.5	0.12-0.5	NA
Linezolid	2	2	0.5-2	0
Quinupristin-dalfopristin	0.25	0.5	0.12-0.5	0
Penicillin	≤0.03	≤0.03	≤0.03-0.06	0
Cefotaximea	≤0.03	≤0.03	≤0.03-0.25	0
Erythromycin	0.06	0.12	≤0.03-0.12	0
Clindamycin	0.06	0.12	≤0.03-0.12	0
Levofloxacin	1	1	0.5-2	0

^aCefotaxime MICs were analyzed with the current NCCLS meningitis breakpoints (22).

^bNA, not applicable.

The calcium content of the test media included in this study varied widely. Although the target value of calcium for the reference broth microdilution method was 50 μg/ml, the final medium contained a measured concentration of 44 μg/ml. One of the agar lots had the desired content of 50 μg/ml, although three lots from a different manufacturer contained only 11 to 14 μg/ml (Tables 2 and 3). The differing calcium contents of the Mueller-Hinton sheep blood agars examined in this study had a significant effect on the daptomycin MICs by the E test method (Table 2). The daptomycin E strips without calcium provided the best correlation with MICs determined by the NCCLS broth microdilution method when applied to the BD Mueller-Hinton agar (calcium content, 50 μg/ml), i.e., 90.9% essential agreement of MICs. E strips that contained calcium plus daptomycin yielded MICs fourfold or more lower when used with BD Mueller-Hinton agar, which resulted in an essential agreement of only 63.1% with the reference broth microdilution MICs. Conversely, the daptomycin E strips that included calcium provided the best correlation with MICs determined by the NCCLS broth microdilution method when applied to the Remel Mueller-Hinton agar that was largely deficient in calcium (11 to 14 μg/ml), i.e., 96% essential agreement of MICs. Daptomycin MICs with the non-calcium-supplemented E strips provided much higher MICs when the Remel Mueller-Hinton agar was used, resulting in 0% essential agreement. Of note, the daptomycin MICs generated by the broth microdilution reference method and both E test formulations and all Mueller-Hinton agar lots were always within the 4-dilution approved NCCLS range (0.06 to 0.5 μg/ml) with S. pneumoniae ATCC 49619 (22).

TABLE 2.

Comparison of daptomycin E test MICs and NCCLS broth microdilution MICs according to the Mueller-Hinton agar test medium and E test formulation

Type of M-Hb agar (calcium concn [μg/ml])	E test format	n	No. of isolates with MIC differencec:										% EAa
			−4	−3	−2	−1	Same	+1	+2	+3	+4	+5
BD (50)	Daptod only	350	0	0	1	5	67	246	29	0	2	0	90.9
BD (50)	Dapto + Ca	350	1	10	118	198	22	1	0	0			63.1
Remel (11-14)	Dapto only	50	0	0	0	0	0	0	0	22	26	2	0
Remel (11-14)	Dapto + Ca	50	0	0	2	33	15	0	0	0	0	0	96

^aEA, essential agreement (percentage of MICs within 1 log₂ dilution of the reference MIC).

^bM-H, Mueller-Hinton.

^cDifference values are log₂ dilutions. Negative values indicate that the E test MIC is lower than the broth microdilution MIC.

^dDapto, daptomycin.

TABLE 3.

Comparison of daptomycin zone diameters according to the Mueller-Hinton agar brand and its calcium content

M-Ha agar	Calcium content (μg/ml)	n	Mean daptomycin zone diam (mm)	Range of zone diams (mm)
BD	50	350	23	20-27
Remel	11-14	50	21	19-24

^aM-H, Mueller-Hinton.

The calcium content also affected daptomycin zone diameters when determined by the NCCLS disk diffusion test, but to only a modest degree (∼2 mm). Since zone size interpretive criteria for daptomycin have not yet been established, it is not possible to determine if the smaller zones developed on the Mueller-Hinton agar that contained suboptimal amounts of calcium would result in interpretive errors. Nevertheless, daptomycin disk zones seemed less affected by the calcium content of the media than MICs generated by E tests on the same medium lots. Routine quality control testing of S. pneumoniae ATCC 49619 did not reveal any deficiencies in the performance of the Mueller-Hinton sheep blood agar lots.

While earlier studies that examined the activity of daptomycin against pneumococci focused only on penicillin-susceptible and nonsusceptible strains (2, 3, 23, 24, 26), we assessed its activity against strains with resistance to several other drug classes (i.e., macrolides, lincosamides, fluoroquinolones, and quinupristin-dalfopristin), Based on our findings and those of others (2, 3, 10, 11, 17, 23, 24, 26) it appears that daptomycin should be considered, along with vancomycin and linezolid, an agent with predictable activity against contemporary pneumococci.

The calcium content of susceptibility test media has been shown previously to affect daptomycin susceptibility results (3, 10, 26) due to the calcium-dependent nature of the drug's binding to its molecular target (19). The Mueller-Hinton sheep blood agars examined in this study had a significant effect on the daptomycin MICs determined by the E test method. Thus, a laboratory would need to carefully match the E test formulation to the calcium content of their test media. Alternatively, commercial medium manufacturers could strive to provide media with approximately the target concentration of calcium so that the standard daptomycin E test strips could be used with confidence. For unclear reasons, the calcium content of the agars did not seem to result in a large variance in zone diameters by the standard NCCLS disk test. Unfortunately, routine quality control testing with the NCCLS-designated strain S. pneumoniae ATCC 49619 did not reveal any deficiencies in the various Mueller-Hinton agar lots. Diagnostic product manufacturers should strive to achieve standardization of test reagents and media so that this promising new drug can be tested reliably by clinical microbiology laboratories if clinical studies lead to the eventual approval of daptomycin for clinical use.