A Time–Kill Evaluation of Clarithromycin and Azithromycin against Two Extracellular Pathogens and the Development of Resistance

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Abstract
OBJECTIVE: To determine the activity of clarithromycin, its metabolite (14-hydroxyclarithromycin), and azithromycin against Haemophilus influenzae and Staphylococcus aureus using time–kill methodology and to evaluate the susceptibility of the organisms following exposure to various concentrations of the azalide macrolides. DATA SOURCES AND METHODS: Clinical isolates of H. influenzae and S. aureus were obtained from the Clinical Microbiology Laboratory at University Hospital, San Antonio, Texas. Susceptibility testing was performed according to National Committee for Clinical Laboratory Standards guidelines. 14-Hydroxyclarithromycin was added to clarithromycin solutions used for H. influenzae. Time–kill studies were performed using antimicrobial concentrations of 0.25–8 x minimum inhibitory concentration (MIC) and an initial inoculum of ∼105 CFU/mL. Samples were plated onto solid agar at 0, 4, 8, 12, and 24 hours. At 0, 12, and 24 hours, samples were then plated onto solid agar incorporated with antibiotic. After incubating plates at 35 °C for 24 hours, colony counts were determined. RESULTS: The MICs of clarithromycin and clarithromycin plus 14-hydroxyclarithromycin for H. influenzae were 4 and 2 μg/mL, respectively. For S. aureus, the MIC of clarithromycin was 0.25 μg/mL, and the MIC of azithromycin for both organisms was 1 μg/mL. H. influenzae developed resistance to both macrolides within 12 hours when exposed to sub-MICs of clarithromycin plus 14-hydroxyclarithromycin. However, when exposed to concentrations less than or equal to the MIC of azithromycin, resistance was not conferred to clarithromycin. S. aureus, on the other hand, became resistant to azithromycin and less susceptible to clarithromycin following exposure to sub-MICs of either macrolide. CONCLUSIONS: Clarithromycin and azithromycin elicited a concentration-independent bacteriostatic effect against H. influenzae and S. aureus at concentrations at least two times the MIC. In addition, concentrations maintained above the MIC prevented changes in the susceptibility of H. influenzae and S. aureus to both macrolides.