In vivo transposition of insertion sequence ISAba1 at the origin of genome plasticity and acquired antibiotic resistance in Acinetobacter baumannii
Abstract number: O295
Poirel L., Mugnier P., Nordmann P.
Background: Insertion sequence ISAba1 belonging to the IS4 family has been identified repeatedly in Acinetobacter baumannii, being often associated with antibiotic resistance genes. In particular, it has been shown to provide promoter sequences enhancing expression of the natural cephalosporinase AmpC and oxacillinase OXA-51 of A. baumannii. In addition, two copies of ISAba1 bracketing blaOXA-23 may form composite transposon Tn2006 at the origin of acquisition of this carbapenem-hydrolysing oxacillinase gene. The transposase of ISAba1 is formed by two open reading frames Orf1 and Orf2 likely giving rise to a functional transposase when a frameshift occurs. Our study was aimed to evaluate the transposition ability of ISAba1.
Methods: We evaluated experimentally the ability of ISAba1 to transpose in Escherichia coli. In a first step, the b-lactamase gene blaTEM-1 was inserted into ISAba1 to provide an ampicillin resistance marker allowing its tracing. This ISAba1::blaTEM-1 structure was cloned into pTOPO vector and then transformed into recA(-) E. coli RZ201 harboring conjugative plasmid pOX38-Gen used as a target for transposition events. Transposition events onto pOX38-Gen were selected by conjugating this plasmid into azide-resistant E. coli J53. The same technique was used to evaluate the transposition ability of Tn2006. Also the role of the frameshift in the transposase expression was evaluated by eliminating the frameshift in the transposase encoding gene by site-directed mutagenesis.
Results: Transposition of ISAba1 was obtained at a frequency of 1×105 (±0.7×105) per E. coli donor. Sequencing of the target sites of transposition revealed i) a systematic 9-bp duplication upon transposition, and an adenine-rich hotspot of target transposition. Transposition frequency of the mutated ISAba1 with a transposase gene made of a unique frame was 2×103 (±0.2×103). The mobility of Tn2006 was demonstrated and its frequency of transposition was estimated to be 1.6×108 (±2.5×102).
Conclusion: This study iss the very first demonstration of the functionality of the widespread ISAba1 as a mobile element. The frameshift-mediated down-regulation of transposition has been assessed, as well as the functionality of the ISAba1-made composite transposon. This in-vivo model will allow further experiments able to evaluate the possible selective factors such as antibiotics for ISAba1-transposition enhance
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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