Precision of vancomycin and daptomycin MICs for methicillin-resistant Staphylococcus aureus and effect of subculture and storage

Abstract

The reproducibility of vancomycin and daptomycin MICs, measured by broth microdilution (BMD) and Etest, was prospectively assessed for 10 methicillin-resistant Staphylococcus aureus (MRSA) isolates from the blood samples from patients on vancomycin therapy. The isolates were tested at the time of isolation from blood and following 5, 10, and 20 subcultures and at 1, 3, 6, and 12 months of storage at -70 °C. The MICs were determined by Etest and BMD using two different manufacturers (BBL and Difco) of cation-adjusted Mueller-Hinton broth (CA-MHB), and using three different drug powders: vancomycin from Sigma, vancomycin from Novation, and daptomycin from Cubist. The antimicrobial concentrations tested were 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 μg/ml. Two isolates were vancomycin intermediate and daptomycin nonsusceptible, and two isolates had reduced susceptibility to vancomycin (BMD MIC, 1.5 or 2.0 μg/ml). The vancomycin MICs were significantly higher in the BBL CA-MHB than those in the Difco CA-MHB, and with Sigma versus Novation vancomycin powder. The daptomycin MICs were also significantly higher in the BBL CA-MHB. The Etest MICs were significantly higher than those obtained by BMD for vancomycin but not for daptomycin. The average precision of the vancomycin BMD MICs when analyzing 20 results was ± 1.10-fold log2 dilutions, and it was ± 1.67-fold for daptomycin (10 results). The average precision for Etest was ± 1.11-fold for vancomycin and ± 1.16-fold for daptomycin. No significant change in MICs was noted following 5, 10, or 20 subcultures or at up to 6 months of frozen storage. However, the vancomycin MICs alone were significantly lower (0.74-fold) following 12 months of frozen storage. From these data, despite variations in CA-MHB and antimicrobial powder, the MIC result precision was <0.5 log2 dilutions in a single laboratory, suggesting that testing interdilution MICs (e.g., MICs between serial 2-fold dilutions) is a possibility. A more accurate method for measuring vancomycin MIC results is thus possible, but further standardization of BMD testing would be required to achieve this goal.

FIG 1

Mean BMD results (n = 20) for 5 isolates tested on day 1. Error bars represent 95% confidence intervals.

FIG 2

Effect of test method on MIC. Shown are the mean vancomycin (filled circles) and daptomycin (open circles) MICs from testing 10 MRSA strains at the time of isolation. Error bars represent 95% confidence intervals.

FIG 3

Effect of subculture and storage on MIC. Shown are the mean vancomycin (filled circles) and daptomycin (open circles) MICs obtained from testing 10 MRSA strains at the time of isolation and following subculture/freezing.