Susceptibility of Enterobacter to Cefamandole: Evidence for a High Mutation Rate to Resistance

Abstract

Cefamandole minimum inhibitory concentrations (MICs) of 10 strains of Enterobacter were determined by the ICS agar dilution and broth dilution procedures. Agar dilution MICs ranged from 1 to 8 μg/ml, with an inoculum of 10⁴ organisms/spot. Broth dilution MICs were consistently higher, with an inoculum of approximately 7 × 10⁵ organisms/ml. Seven strains showed MICs of ≥64 μg/ml. There was a marked inoculum effect in broth, and skipped tubes were often observed. Variants resistant to 32 μg/ml or more were isolated by direct selection and were shown to occur at a frequency of approximately 10⁻⁶ to 10⁻⁷. A mutant showing a 16-fold increase in agar dilution MIC was also isolated by indirect selection. These variants and others isolated from broth in the presence of cefamandole were tested for ability to inactivate the antibiotic, using both a biological and a chemical procedure. Two distinct classes of variants were seen. Twelve of 28 were shown by both methods to inactivate the antibiotic, whereas the others, including the indirectly selected mutant, did not. The wild types were also negative by both tests. The higher cefamandole MICs of Enterobacter in broth, thus, appeared to reflect a high frequency of resistant variants that were not detected with the inoculum and end point criteria usually used in agar dilution methods. The ability of some variants to inactivate cefamandole may have resulted from a mutation that extended the activity of Enterobacter cephalosporinase to include this antibiotic.