Construction of a probe for the aminoglycoside 3-V-acetyltransferase gene and detection of the gene among endemic clinical isolates

Abstract

A recent surveillance study at Vanderbilt University Medical Center indicated that 8.5% of the gram-negative bacilli isolated were resistant to gentamicin. To determine what proportion of current gentamicin-resistant isolates elaborated aminoglycoside 3-V-acetyltransferase [AAC(3)-V], a probe for this gene was constructed from a 975-base-pair PstI-SalI fragment of the nonconjugative R plasmid pCER954b. This plasmid was first isolated at Vanderbilt University Medical Center from epidemic strains of Serratia marcescens. Nineteen isolates determined to produce AAC(3)-V by MIC profile all reacted with the probe. The probe did not hybridize with DNA from organisms producing 10 other aminoglycoside-modifying enzyme types. With this probe, 30 (36%) of 84 gentamicin-resistant, gram-negative bacilli elaborated AAC(3)-V. Of these 30 isolates, 25 contained a conjugative plasmid that transferred gentamicin resistance. In contrast to other medical centers, at Vanderbilt a sizable number of gentamicin-resistant, gram-negative bacilli produced AAC(3)-V. This resistance determinant, initially identified in an epidemic Serratia strain, has persisted and become incorporated into currently isolated endemic strains of gram-negative bacilli.