Identification of individual aminoglycoside-inactivating enzymes in a mixture by HPLC determination of reaction products.

A method is described for the detection and identification of two aminoglycoside-acetylating (AAC(2') or AAC(3) or AAC(6')) or -phosphorylating (APH(2'') or APH(3')) enzymes when these are present in the same crude enzyme extract. Crude enzyme extracts were prepared from reference strains of bacteria producing AAC(2'), AAC(3), AAC(6'), APH(2'') or APH(3') enzymes and mixed to give the following paired enzyme combinations: AAC(2') + AAC(3), AAC(2') + AAC(6'), AAC(3) + AAC(6') and APH(2") + APH(3'). These enzyme mixtures were examined by the radioenzymatic profile method for the identification of aminoglycoside-modifying enzymes and their reaction products with butirosin, lividomycin and kanamycin (AAC) or lividomycin and kanamycin (APH) characterized by high performance liquid chromatography (HPLC). Only two of the eight enzymes present were identified by the radioenzymatic method, but all eight were correctly identified by the HPLC method.