High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3′-phosphotransferase with high affinity for amikacin

Abstract

This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3′)-VI. The enzyme from P. aeruginosa PA40 was purified to >70% homogeneity. The K m of amikacin for this enzyme was 1.4 μM, the V max/ K m ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3′ )-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3′)-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed.