Inhibition of AAC(6')-Ib-mediated resistance to amikacin in Acinetobacter baumannii by an antisense peptide-conjugated 2',4'-bridged nucleic acid-NC-DNA hybrid oligomer

Abstract

Multiresistant Acinetobacter baumannii, a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors aac(6')-Ib. This gene specifies resistance to amikacin and other aminoglycosides, seriously limiting the effectiveness of these antibiotics. An antisense oligodeoxynucleotide (ODN4) that binds to a duplicated sequence on the aac(6')-Ib mRNA, one of the copies overlapping the initiation codon, efficiently inhibited translation in vitro. An isosequential nuclease-resistant hybrid oligomer composed of 2',4'-bridged nucleic acid-NC (BNA(NC)) residues and deoxynucleotides (BNA(NC)-DNA) conjugated to the permeabilizing peptide (RXR)4XB ("X" and "B" stand for 6-aminohexanoic acid and β-alanine, respectively) (CPPBD4) inhibited translation in vitro at the same levels observed in testing ODN4. Furthermore, CPPBD4 in combination with amikacin inhibited growth of a clinical A. baumannii strain harboring aac(6')-Ib in liquid cultures, and when both compounds were used as combination therapy to treat infected Galleria mellonella organisms, survival was comparable to that seen with uninfected controls.

FIG 1

Chemical structure of a 2′,4′-BNA^NC residue.

FIG 2

Nucleotide sequences targeted by antisense ODNs. The nucleotide sequences around the initiation codon of the A. baumannii (Ab) A155 aac(6′)-Ib gene are shown at the bottom of the figure. The duplicated sequences are within red and blue boxes. The sequences of the ODNs tested and the locations of antisense regions are shown at the top.

FIG 3

In vitro activities of ODNs. Cell-free translation reactions were carried out as described in Materials and Methods in the absence (−) or presence (+) of the indicated ODNs at 6.6 μM. The products were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the radioactivity was detected with a phosphorimager. A control lacking aac(6′)-Ib mRNA was included (−). (A) “AS” stands for “antisense,” and the numbers indicate the ODN numbers corresponding to those shown in Fig. 2. “P” and “S” stand for ODNs antisense to a phoA sequence and sense sequence, respectively. (B) “AS” stands for “antisense.” “C4” and “CP” stand for “CPPBD4” and “CPPBDAP,” respectively.

FIG 4

Effect of CPPBD4 on resistance to AMK. A. baumannii A155 was cultured in 100 μl Mueller-Hinton broth in microtiter plates at 37°C, with the additions indicated in the figure, and the OD₆₀₀ was determined every 20 min. CPPBD compounds were added at 0.5 μM and AMK at 4 μg/ml.

FIG 5

G. mellonella infection and treatment assays. Final-instar larva groups of 10 individuals were injected with the components shown in the figure, and a control group was not injected (“No injection”). The concentrations of the injected components were 10 mg AMK/kg of body weight and 0.5 μM CPPBD4 and CPPBDAP. The larvae were incubated at 37°C in the dark, and survival was recorded at 24-h intervals over 120 h.