ISAba825 as a modulator of genome plasticity and plasmid-borne bla(OXA-58) expression in Acinetobacter baumannii

VIALE A.M.; RAVASI, P.; LIMANSKY, A.S.; MUSSI M.A.

Bariloche

Congreso; International Plasmid Biology Conference 2010; 2010

IPCB

Background and aims:  The emerging appearance of carbapenem resistance among multidrug-resistant (MDR) Acinetobacter baumannii nosocomial strains represents a major concern worldwide. Mechanisms proposed to play significant roles in A. baumannii carbapenem resistance are the overproduction of acquired OXA-type carbapenemases, and alterations in outer membrane (OM) permeability. We recently reported that the loss of the OM channel CarO as a result of the insertional inactivation of its coding gene by a novel insertion sequence, ISAba825 (1). Since IS can induce genome rearrangements resulting in increased antimicrobial resistance, we evaluated ISAba825 functionality and impact on the plasticity of the A. baumannii genome. We report here that ISAba825 insertion can increase expression of plasmid-borne blaOXA-58 in MDR clinical strains of A. baumannii, resulting in the acquisition of additional carbapenem-resistant phenotypes. Methods: We evaluated the host transposition capability range of ISAba825 by tagging this element with a kanamycin resistance cassette generating ISAba825::Kn in a suicide plasmid, pknock825, and transforming this plasmid into A. baumannii ATCC 17978, E. coli DH5a, and Acinetobacter baylyi. Transposition was evaluated by selection of Kn-resistant clones, PCR, and sequencing analyses. Whether ISAba825 is located in close proximity to blaOXA genes in carbapenem-resistant A. baumannii clinical strains isolated in Rosario hospitals was evaluated by PCR using appropriate primer combinations for ISAba825 and different blaOXA genes followed by sequencing analyses. The presence of ISAba825-blaOXA-58 in a plasmid location was determined by Southern analysis, and the ability of this plasmid to significantly increase blaOXA-58 expression was analyzed by both 5´-RACE and by measuring the MICs for imipenem and meropenem in transformed A. baumannii ATCC 17978. Results: ISAba825::Kn could transpose only in A. baumannii, indicating an extremely narrow species range. In one carbapenem-resistant A. baumannii clinical strain, Ab880, we found ISAba825 truncating the tnpA gene of an ISAba3-like element located upstream of a blaOXA-58 gene, all elements present in an indigenous plasmid, pAb880. RACE analysis indicated that ISAba825 insertion generated an alternative hybrid promoter for blaOXA-58 expression, resulting in a 16 to 32-fold increase in MICs to carbapenems in pAb880-containing A. baumannii. Concerning ISAba825 distribution, PCR analysis indicated its presence only in carbapenem-resistant isolates of our collection. Conclusions: The prevalence of ISAba825 in carbapenem-resistant strains enhancing blaOXA-58 expression, in addition to its inactivation of carO reported previously (1), lead us to propose a role for this IS in the modulation of carbapenem resistance in A. baumannii. Also, this is the first report characterizing the transposition of an IS using A. baumannii as a host. (1) Mussi et al. (2005) Antimicrobial Agents Chemother. 49: 1432-1440