Original article
Daptomycin susceptibility tests: interpretive criteria, quality control, and effect of calcium on in vitro tests

A portion of this material was previously presented at the 39th ICAAC in San Francisco, California, September 26–29, 1999. Abstract No. 350.
https://doi.org/10.1016/S0732-8893(00)00164-4Get rights and content

Abstract

Daptomycin MICs were determined for 844 Gram-positive bacteria in three concentrations of Ca++ and compared with the MICs of vancomycin and teicoplanin. Daptomycin was twofold to fourfold more active against most species when tested in 50 μg/ml of Ca++ than in 25 μg/ml. In 50 μg/ml of Ca++ daptomycin was more active against methicillin-resistant staphylococci and vancomycin-resistant enterococci than teicoplanin or vancomycin; 100% of these isolates were susceptible to ≤2.0 μg/ml of daptomycin. Different lots of Mueller-Hinton agar were variable in Ca++ content, and daptomycin disk diffusion zone diameters were affected, i.e., zones were 1 to 15 mm smaller on one lot of agar with only 6 μg/ml of Ca++ compared to another lot with 28 μg/ml. The previously proposed daptomycin interpretive breakpoints performed satisfactorily when MICs were determined in Mueller-Hinton broth with 50 μg/ml of Ca++ and when the agar gave appropriate zones with quality control strains. To define those control limits, replicate tests with four quality control strains were performed in ten laboratories using broth microdilution tests (with Ca++ supplemented broth) and disk diffusion tests on Mueller-Hinton agar without cation adjustments.

Introduction

Daptomycin is a cyclic lipopeptide antibiotic that has been reported to have good antimicrobial activity against most Gram-positive bacteria Appelbaum et al 1989, Eliopoulos et al 1986, Greenwood and Palfreyman 1987, Low et al 1989, Pohlod et al 1987, Swenson et al 1990. Despite encouraging results with daptomycin in experimental animals Bryant et al 1987, Cantoni et al 1990, Ramos et al 1992, further development of the drug was suspended after early clinical trials showed that relatively low doses of daptomycin (total of 2 mg/kg/day) were less effective than conventional therapy (Chambers, 1991) and high doses of 4 mg/kg q12h (8 mg/kg/day) produced muscle toxicity in a few patients. In animal studies this muscle toxicity has been shown to be significantly reduced by once-daily dosing (Oleson et al., 1999). Furthermore, since the Cmax has been shown to be the major pharmacokinetic parameter associated with infection eradication in animal efficacy models (Leggett et al., 1987), once-daily dosing could be more effective. With the increasing prevalence of vancomycin-resistant enterococci (VRE), methicillin-resistant staphylococci (MRS) and penicillin-resistant pneumococci (PRP), there has been renewed interest in developing daptomycin for clinical use. Daptomycin Phase II trials using once-daily dosing are currently in progress with encouraging preliminary safety results (Tally et al., 1999).

The in vitro activity of daptomycin is enhanced by increased concentrations of Ca++ when tested in broth Eliopoulos et al 1986, Greenwood and Palfreyman 1987, Louie et al 1992. The interpretive criteria for daptomycin that were proposed in 1987 (Jones & Barry 1987) were based on MIC tests performed in cation-supplemented Mueller-Hinton broth (CSMHB) containing 50 μg/ml of Ca++, as recommended at that time by the National Committee for Clinical Laboratory Standards (NCCLS) (1985). Since then the NCCLS has revised its recommendations, and currently recommends the use of cation-adjusted Mueller-Hinton broth (CAMHB) containing 20–25 μg/ml of Ca++ for routine susceptibility testing (NCCLS 1997b).(Jones 1989) suggested that when testing staphylococci, daptomycin MICs were higher when CAMHB was used, but the interpretive criteria developed in CSMHB could still be applied as all isolates were susceptible in both concentrations of Ca++. Enterococci were not a concern when that study was undertaken and thus only staphylococci were evaluated.

The present studies were designed to: 1) Confirm the effects of Ca++ on in vitro activity of daptomycin against Gram-positive bacteria including enterococci, 2) Compare the activity of daptomycin to that of vancomycin and teicoplanin, 3) Assess the daptomycin interpretive criteria in light of the current recommendation to use CAMHB (ca. 25 μg/ml Ca++) instead of CSMHB (ca. 50 μg/ml Ca++) for MIC determinations, and 4) Determine the daptomycin quality control (QC) ranges that can be used to evaluate the suitability of Mueller-Hinton broth or agar lots for testing daptomycin.

Section snippets

Microorganisms

A total of 844 Gram-positive bacterial isolates, representing 14 species were tested (Table 1 A large proportion of antibiotic-resistant strains was selected, including 50 VRE, 147 MRS, 52 PRP, and 111 penicillin-intermediate pneumococci. On each day of testing appropriate QC strains were tested and all results were within the published NCCLS QC ranges (NCCLS, 1999).

Antimicrobial Agents

Daptomycin was provided by Cubist Pharmaceuticals, Inc., Cambridge, MA. The comparison drugs were procured from their U.S.

Effect of Calcium

The effect of three concentrations of Ca++ on daptomyin MICs is summarized in Table 1. Against all species daptomycin was significantly more active in 50 μg/ml than in 25 μg/ml of Ca++. When tested in 75 μg/ml of Ca++ there was a further increase in daptomycin activity for some species, but this was generally much less than the differences between 25 and 50 μg/ml of Ca++. For other species there was no increase in activity when tested in 75 μg/ml of Ca++ (e.g., streptococci). If one accepts the

Discussion

The effect of Ca++ on daptomycin broth dilution MICs has been well-documented previously Eliopoulos et al 1986, Greenwood and Palfreyman 1987, Louie et al 1992, and our data confirm that daptomycin MICs are generally twofold to fourfold lower when tested in broth containing 50 μg/ml of Ca++ than when tested in 25 μg/ml of Ca++. To our knowledge, it has not been previously demonstrated that Ca++ levels in MHA can significantly affect the daptomycin disk diffusion zone diameters. In this study,

Acknowledgments

We gratefully acknowledge the participation of the following in the ten-laboratory quality control study: Mary Bauman, St. Vincent’s Hospital, Portland, OR; Mary Jane Ferraro, Massachusetts General Hospital, Boston, MA; Dwight Hardy, University of Rochester Medical Center, Rochester, NY; Janet Hindler, UCLA Medical Center, Los Angeles, CA; Stephen Jenkins, Carolinas Medical Center, Charlotte, NC; Cindy Knapp, AccuMed International, Westlake, OH; Gary Overturf, University of New Mexico Medical

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