Multicopy blaOXA-58 gene as a source of high-level resistance to carbapenems in Acinetobacter baumannii

Abstract

The mechanisms at the origin of heterogeneous carbapenem resistance levels observed among Acinetobacter baumannii isolates collected in 2005 in a large University Hospital of Rome, Italy, were investigated. These isolates were related and possessed similar plasmids carrying the carbapenem-hydrolyzing oxacillinase gene bla(OXA-58) but showed variable levels of resistance to carbapenems. Analysis of sequences surrounding the bla(OXA-58) gene showed genetic variability, with the presence in several isolates of multiple copies of the bla(OXA-58) gene; this extra copy number was likely related to an IS26-mediated transposition or recombination process.

FIG. 1.

Plasmid localization of the bla_OXA-58 gene. Shown is agarose (1%) gel electrophoresis in 1× Tris-acetate-EDTA buffer of SacI- or HindIII-digested plasmids from A. baumannii isolates, stained with ethidium bromide and visualized under UV light, and Southern blot hybridization with the bla_OXA-58 probe. M is a 1-kb Plus Ladder, and M₁ is the HindIII-digested lambda DNA (Invitrogen, Milan, Italy).

FIG. 2.

Consensus map of the bla_OXA-58 gene region. The insertion sequences identified within the resistance regions are shown as boxes, and the arrows indicate the directions of transcription of the genes. The bla_OXA-58 gene is represented by the heavy black arrow. Restriction sites for EcoRI, HindIII, and SacI are indicated. The bars below the map represent the positions of the constructs obtained from the libraries and sequenced. The dashed lines show the insertion site of the second copy of the IS26-ISAba2-bla_OXA-58-ISAba3 module in isolate 193 with respect to isolate 183. The brackets indicate the presence of two IS26-ISAba2-bla_OXA-58-ISAba3 modules in isolate 186.

FIG. 3.

Determination of the bla_OXA-58 gene copy number by real-time PCR amplification. Shown are amplification curves obtained in quantitative real-time PCR experiments performed on the repAci1 gene (one copy per plasmid) and the bla_OXA-58 gene measured for the isolates 183 (one copy per plasmid), 193 (two copies per plasmid), and 186 (three copies per plasmid).