Regrowth of aminoglycoside-resistant variants and its possible implication for determination of MICs

Regrowth of aminoglycoside-resistant variants was seen when large inocula of two strains of Escherichia coli were incubated with gentamicin in concentrations well above their MICs (0.5 micrograms/ml). The extent of the selection of resistant variants was proportional to the concentration of gentamicin during incubation; after incubation with gentamicin (greater than or equal to 2 micrograms/ml for 24 h), all bacteria were resistant to at least 8 micrograms/ml. Bacteria resistant to these concentrations always formed small colonies, whereas variants resistant to lower concentrations (1 to 2 micrograms/ml) could form both small and normal colonies. The regrowth of resistant variants could be monitored by luciferase assay of intracellular ATP in cultures incubated with gentamicin (less than or equal to 2 micrograms/ml). In cultures incubated with higher concentrations, regrowth did occur, although this did not result in viability (CFU per milliliter) or ATP levels above those of the initial inocula. The implications of this regrowth for MIC determinations in broth and the possible clinical revelance of the resistant variants are discussed.