A new simple assay for determining aminoglycoside inactivation in intact cells of Pseudomonas aeruginosa.

Intact cells of aminoglycoside (AG) antibiotic-resistant Pseudomonas aeruginosa usually do not inactivate AG, even though they possess the AG-modifying enzyme. An assay method for determining the activity of inactivating enzyme in intact cells of streptomycin-resistant P. aeruginosa was previously reported. Although this assay method was applied to the determination of the activity of kanamycin (KM)-inactivating enzymes, it could not apply to some of the KM-resistant strains. A new simple assay method has now been investigated for determining the activity of KM-inactivating enzyme in intact cells of clinically isolated KM-resistant P. aeruginosa. The determination of AG-inactivating enzyme activity was attempted using lysozyme for release of the inactivating enzymes in washed cells, and both DNase and RNase were added to digestion of the nucleic acids released by bacteriolysis. This lysozyme-DNase-RNase (LDR) method has facilitated the confirmation of the presence of AG-inactivating enzyme in the strains used. In addition, the LDR technique was applicable to the determination of inactivating enzyme activity for various AGs other than KM. Since this simple assay method can determine any type of AG-inactivating enzyme activity of various P. aeruginosa strains, it may contribute significantly to the rapid selection of drugs in clinical use.