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Quantitative Assessment of Bactericidal Activities of β-Lactam Antibiotics by Agar Plate Method

Quantitative bactericidal activities of β-lactam antibiotics were determined by the agar plate method. Broth cultures, of which the colony-forming units were counted before the study, were inoculated on antibiotic-containing agar plates, utilizing a 10−3, 10−2, or 10−1 dilution or undiluted culture plated with each 0.001-ml calibrated loop. These plates were incubated at 37°C overnight, and the minimal drug concentration at which no bacterial growth was observed on the plates was defined as minimal inhibitory concentration. After this procedure, the agar surface was treated with β-lactamase spray to inactivate the antibiotic. These plates were incubated again at 37°C overnight. The minimal drug concentration at which no evidence of bacterial growth was visible on the plates (resulting in a 100% kill) was defined as minimal bactericidal concentration. The lowest concentration which reduced the number of colony-forming units to 1/1,000 that in the original inoculum (resulting in a 99.9% kill) was defined as minimal lethal concentration. When compared for Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, alpha-hemolytic streptococcus (non-enterococcal), beta-hemolytic streptococcus, and enterococcus, the minimal bactericidal concentrations were generally several fold higher than the minimal inhibitory concentrations. Minimal lethal concentrations were virtually the same as minimal inhibitory concentrations for gram-negative strains; however, for some gram-positive strains, minimal lethal concentrations were higher than minimal inhibitory concentrations.