Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6 )-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6 )-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nucleaseresistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6 )-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.aminoglycoside ͉ antibiotic resistance ͉ antisense ͉ nucleic acids analogs ͉ RNase P

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.

The dissemination of AAC(6)-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase Pmediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6)-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6)-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6)-Ib, and the transformant strains were tested to determine their resistance to amikacin. The two external guide sequences that showed the strongest binding efficiency to the mRNA in vitro, EGSC3 and EGSA2, interfered with expression of the resistance phenotype at different degrees. Growth curve experiments showed that E. coli cells harboring a plasmid coding for EGSC3, the external guide sequence with the highest mRNA binding affinity in vitro, did not grow for at least 300 min in the presence of 15 g of amikacin/ml. EGSA2, which had a lower mRNA-binding affinity in vitro than EGSC3, inhibited the expression of amikacin resistance at a lesser level; growth of E. coli harboring a plasmid coding for EGSA2, in the presence of 15 g of amikacin/ml was undetectable for 200 min but reached an optical density at 600 nm of 0.5 after 5 h of incubation. Our results indicate that the use of external guide sequences could be a viable strategy to preserve the efficacy of amikacin.Aminoglycoside antibiotics act by interfering with the decoding process (12,22,25,39) and are used to treat infections caused by aerobic gram-negative or gram-positive bacteria. In this latter case they are used in combination with other antibiotics (46). However, the rise in resistant and multiresistant strains, usually harboring aminoglycoside modifying enzymes, has limited the successful use of aminoglycosides in the treatment of serious infections (17, 37). The semisynthetic aminoglycoside amikacin (AMK) has been very useful in the treatment of multiresistant infections because a limited number of modifying enzymes, such as AAC(6Ј)-I-type acetyltransferases, are able to inactivate it (37). However, the spread of these enzymes has rendered AMK all but useless in some regions of the world (6,30,38,45). Development of strategies to inhibit the expression of resistance genes could preserve the efficacy of AMK. Antisense oligonucleotide technologies may be an alternative for this purpose. A variety of antisense strategies have been applied before to inhibit gene expression in prokaryotic systems (4,10,23,28,34,44).RNase...

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