Inhibition of Aminoglycoside 6′- N -Acetyltransferase Type Ib-Mediated Amikacin Resistance by Antisense Oligodeoxynucleotides

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Abstract
Amikacin has been very useful in the treatment of infections caused by multiresistant bacteria because it is refractory to the actions of most modifying enzymes. However, the spread of AAC(6′)-I-type acetyltransferases, enzymes capable of catalyzing inactivation of amikacin, has rendered this antibiotic all but useless in some parts of the world. The aminoglycoside 6′-N-acetyltransferase type Ib, which is coded for by the aac(6′)-Ib gene, mediates resistance to amikacin and other aminoglycosides. RNase H mapping and computer prediction of the secondary structure led to the identification of five regions accessible for interaction with antisense oligodeoxynucleotides in the aac(6′)-Ib mRNA. Oligodeoxynucleotides targeting these regions could bind to native mRNA with different efficiencies and mediated RNase H digestion. Selected oligodeoxynucleotides inhibited AAC(6′)-Ib synthesis in cell-free coupled transcription-translation assays. After their introduction into an Escherichia coli strain harboring aac(6′)-Ib by electroporation, some of these oligodeoxynucleotides decreased the level of resistance to amikacin. Our results indicate that use of antisense compounds could be a viable strategy to preserve the efficacies of existing antibiotics to which bacteria are becoming increasingly resistant.