Identification of the aadB gene coding for the aminoglycoside-2"-O-nucleotidyltransferase, ANT(2"), by means of the polymerase chain reaction.

Abstract

The polymerase chain reaction (PCR) was used to identify the gene encoding the aminoglycoside-2"-O-nucleotidyltransferase, ANT(2"). Two primers, delineating a DNA fragment of 188 bp, and a specific probe within this fragment were constructed, based on the nucleotide sequence of the aadB gene encoding this enzyme. Reference strains producing different aminoglycoside-modifying enzymes were used to evaluate the specificity and the sensitivity of the test. Strains producing the ANT(2") enzyme showed the expected 188 bp DNA fragment after amplification. The oligonucleotide primers did not interact with genes encoding other aminoglycoside-modifying enzymes. Evaluation of a one-step single colony technique demonstrated that it was an acceptable alternative to the classic PCR test. The PCR method was used successfully to detect the presence of the aadB gene in 17 gentamicin-resistant clinical isolates.